Compositions and methods for modulating gene or gene product in cells

ABSTRACT

Disclosed herein are methods for modulating an amount or activity of a gene or a gene product in a cell. The methods herein may comprise contacting a cell with a therapeutic agent assembled with a lipid composition, which lipid composition may comprise a dendrimer or dendron which may comprise one or more degradable diacyl group, in which may result in modulating the amount or activity of the gene or the gene product in the cell. The therapeutic agent modulating a gene or gene product in a cell may be sufficient to treat a disease or disorder in a subject. Further disclosed herein are pharmaceutical compositions, kits, and lipid compositions for modulating an amount or activity of a gene or a gene product in a cell.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates generally to the fields of dendrimers. Inparticular, it relates to dendrimer nanoparticle compositions comprisinga nucleic acid. More particularly, it relates to dendrimer nanoparticlecompositions for the delivery of the nucleic acid. More particularly, itrelates to dendrimer nanoparticle compositions for the delivery of drugsand other excipients.

2. Description of Related Art

Since the discovery of RNAi or other nucleic acid agents and therecognition of their therapeutic potential, there has been a continuoussearch for effective delivery carriers (Whitehead et al., 2009; Kanastyet al., 2013; Akinc et al., 2008; Davis et al., 2010; Love et al., 2010;Siegwart et al., 2011; Jayaraman et al., 2012). Progress has been madewith regard to delivery efficacy of small RNAs to healthy livers, butthe clinically required combination of high potency to tumors and lownormal cell hepatotoxicity is not currently met by existing deliveryvehicles. Unfortunately, all five Phase III human clinical trials ofsmall molecule drugs for hepatocellular carcinoma (HCC) treatment failedwithin the past four years in part because debilitating, late-stageliver dysfunction amplifies drug toxicity (Roberts, L. R., 2008;Scudellari, M., 2014). MicroRNAs (miRNAs) represent a promisingalternative strategy because they can function as tumor suppressors byconcurrently targeting multiple pathways involved in celldifferentiation, proliferation, and survival, but these therapeuticagents require carriers to be effective (Ventura and Jacks, 2009;Kasinski and Slack, 2011; Ling et al., 2013; Cheng et al., 2015). Abalance of potency versus toxicity of the drug carrier is a usefulcriteria particularly in the context of liver cancer where the carrier'sown toxicity can abate the therapeutic effectiveness of the small RNAtherapies.

To achieve this balance of low toxicity and high potency, the influenceof chemical structure by expanding the structural diversity andmolecular size of delivery carriers is useful in achieving atherapeutically effective balance. Dendrimers are monodispersemacromolecules composed of multiple perfectly branched monomers thatemanate radially from a central core. The dendrimers therefore have thesame high degree of molecular uniformity as small molecules and thebroad theoretical space for chemical tuning as polydisperse polymers(Bosman et al., 1999; Frdchet and Tomalia, 2002; Gillies and Frechet,2002; Grayson and Frdchet, 2001). These intrinsic characteristics enabledendrimers to have unique properties (Murat and Grest, 1996; Percec etal., 2010; Duncan and Izzo, 2005) for various biomedical applications(Stiriba et al., 2002; Lee et al., 2005; Wu et al., 2015). In genedelivery, most studies have used the limited number of commercialdendrimers for further chemical modification. (Kang et al., 2005;Taratula et al., 2009; Khan et al., 2014). The expansion of dendrimerapplications therefore depends on the ability to easily tune the size,chemistry, topology, and ultimately, dendrimer physical propertiesthrough chemical synthesis. As such, the development of new dendrimerswhich can act as carriers of nucleic acids and other drugs is clinicallyuseful.

SUMMARY OF THE INVENTION

In some aspects, the present disclosure provides dendrimers of theformula:

Core-(Repeating Unit)_(n)-Terminating Group  (I)

-   wherein the core is linked to the repeating unit by removing one or    more hydrogen atoms from the core and replacing the atom with the    repeating unit and wherein:    -   the core has the formula:

-   -   wherein:        -   X₁ is amino or alkylamino_((C≤12)), dialkylamino_((C≤12)),            heterocycloalkyl_((C≤12)), heteroaryl_((C≤12)), or a            substituted version thereof;        -   R₁ is amino, hydroxy, or mercapto, or alkylamino_((C≤12)),            dialkylamino_((C≤12)), or a substituted version of either of            these groups; and        -   a is 1, 2, 3, 4, 5, or 6; or    -   the core has the formula:

-   -   wherein:        -   X₂ is N(R₅)_(y);            -   R₅ is hydrogen, alkyl_((C≤18)), or substituted                alkyl_((C≤18)); and            -   y is 0, 1, or 2, provided that the sum of y and z is 3;        -   R₂ is amino, hydroxy, or mercapto, or alkylamino_((C≤12)),            dialkylamino_((C≤12)), or a substituted version of either of            these groups;        -   b is 1, 2, 3, 4, 5, or 6; and        -   z is 1, 2, 3; provided that the sum of z and y is 3; or    -   the core has the formula:

-   -   wherein:        -   X₃ is —NR₆—, wherein R₆ is hydrogen, alkyl_((C≤8)), or            substituted alkyl_((C≤8)), —O—, or alkylaminodiyl_((C≤8)),            alkoxydiyl_((C≤8)), arenediyl_((C≤8)),            heteroarenediyl_((C≤8)), heterocycloalkanediyl_((C≤8)), or a            substituted version of any of these groups;        -   R₃ and R₄ are each independently amino, hydroxy, or            mercapto, or alkylamino_((C≤12)), dialkylamino_((C≤12)), or            a substituted version of either of these groups; or a group            of the formula: —(CH₂CH₂N)_(e)(R_(c))R_(d);        -   wherein:            -   e is 1, 2, or 3;            -   R_(c) and R_(d) are each independently hydrogen,                alkyl_((C≤6)), or substituted alkyl_((C≤6));        -   c and d are each independently 1, 2, 3, 4, 5, or 6; or    -   the core is alkylamine_((C≤18)), dialkylamine_((C≤36)),        heterocycloalkane_((C≤12)), or a substituted version of any of        these groups;    -   wherein the repeating unit comprises a degradable diacyl and a        linker;        -   the degradable diacyl group has the formula:

-   -   -   wherein:            -   A₁ and A₂ are each independently —O— or —NR_(a)—,                wherein:                -   R_(a) is hydrogen, alkyl_((C≤6)), or substituted                    alkyl_((C≤6));            -   Y₃ is alkanediyl_((C≤12)), alkenediyl_((C≤12)),                arenediyl_((C≤12)), or a substituted version of any of                these groups; or a group of the formula:

-   -   -   -   wherein:                -   X₃ and X₄ are alkanediyl_((C≤12)),                    alkenediyl_((C≤12)), arenediyl_((C≤12)), or a                    substituted version of any of these groups;                -   Y₅ is a covalent bond, alkanediyl_((C≤12)),                    alkenediyl_((C≤12)), arenediyl_((C≤12)), or a                    substituted version of any of these groups; and            -   R₉ is alkyl_((C≤8)) or substituted alkyl_((C≤8));

        -   the linker group has the formula:

-   -   -   wherein:            -   Y₁ is alkanediyl_((C≤12)), alkenediyl_((C≤12)),                arenediyl_((C≤12)), or a substituted version of any of                these groups; and

    -   wherein when the repeating unit comprises a linker group, then        the linker group is attached to a degradable diacyl group on        both the nitrogen and the sulfur atoms of the linker group,        wherein the first group in the repeating unit is a degradable        diacyl group, wherein for each linker group, the next group        comprises two degradable diacyl groups attached to the nitrogen        atom of the linker group; and wherein n is the number of linker        groups present in the repeating unit; and

    -   the terminating group has the formula:

-   -   wherein:        -   Y₄ is alkanediyl_((C≤18)) or an alkanediyl_((C≤18)) wherein            one or more of the hydrogen atoms on the alkanediyl_((C≤18))            has been replaced with —OH, —F, —Cl, —Br, —I, —SH, —OCH₃,            —OCH₂CH₃, —SCH₃, or —OC(O)CH₃;        -   R₁₀ is hydrogen, carboxy, hydroxy, or        -   aryl_((C≤12)), alkylamino_((C≤12)), dialkylamino_((C≤12)),            N-heterocycloalkyl_((C≤12)),            —C(O)N(R₁₁)-alkanediyl_((C≤6))-heterocycloalkyl_((C≤12)),            —C(O)-alkyl-amino_((C≤12)), —C(O)-dialkylamino_((C≤12)),            —C(O)—N-heterocycloalkyl_((C≤12)), wherein:            -   R₁₁ is hydrogen, alkyl_((C≤6)), or substituted                alkyl_((C≤6));    -   wherein the final degradable diacyl in the chain is attached to        a terminating group;    -   n is 0, 1, 2, 3, 4, 5, or 6;        or a pharmaceutically acceptable salt thereof. In some        embodiments, the structure of the dendrimer is further defined:    -   the core has the formula:

-   -   wherein:        -   X₁ is amino or alkylamino_((C≤12)), dialkylamino_((C≤12)),            heterocycloalkyl_((C≤12)), heteroaryl_((C≤12)), or a            substituted version thereof;        -   R₁ is amino, hydroxy, or mercapto, or alkylamino_((C≤12)),            dialkylamino_((C≤12)), or a substituted version of either of            these groups; and        -   a is 1, 2, 3, 4, 5, or 6; and    -   wherein the repeating unit comprises a degradable diacyl and a        linker;        -   the degradable diacyl group has the formula:

-   -   -   wherein:            -   A₁ and A₂ are each independently —O— or —NR_(a)—,                wherein:                -   R_(a) is hydrogen, alkyl_((C≤6)), or substituted                    alkyl_((C≤6));            -   Y₃ is alkanediyl_((C≤12)), alkenediyl_((C≤12)),                arenediyl_((C≤12)), or a substituted version of any of                these groups; or a group of the formula:

-   -   -   -   wherein:                -   X₃ and X₄ are alkanediyl_((C≤12)),                    alkenediyl_((C≤12)), arenediyl_((C≤12)), or a                    substituted version of any of these groups;                -   Y₅ is a covalent bond, alkanediyl_((C≤12)),                    alkenediyl_((C≤12)), arenediyl_((C≤12)), or a                    substituted version of any of these groups; and            -   R₉ is alkyl_((C≤8)) or substituted alkyl_((C≤8));

        -   the linker group has the formula:

-   -   -   wherein:            -   Y₁ is alkanediyl_((C≤12)), alkenediyl_((C≤12)),                arenediyl_((C≤12)), or a substituted version of any of                these groups; and

    -   wherein when the repeating unit comprises a linker group, then        the linker group is attached to a degradable diacyl group on        both the nitrogen and the sulfur atoms of the linker group,        wherein the first group in the repeating unit is a degradable        diacyl group, wherein for each linker group, the next group        comprises two degradable diacyl groups attached to the nitrogen        atom of the linker group; and wherein n is the number of linker        groups present in the repeating unit; and

    -   the terminating group, wherein the terminating group has the        formula:

-   -   wherein:        -   Y₄ is alkanediyl_((C≤18)) or an alkanediyl_((C≤18)) wherein            one or more of the hydrogen atoms on the alkanediyl_((C≤18))            has been replaced with —OH, —F, —Cl, —Br, —I, —SH, —OCH₃,            —OCH₂CH₃, —SCH₃, or —OC(O)CH₃;        -   R₁₀ is hydrogen, carboxy, hydroxy, or        -   aryl_((C≤12)), alkylamino_((C≤12)), dialkylamino_((C≤12)),            N-heterocycloalkyl_((C≤12)),            —C(O)N(R₁₁)-alkanediyl_((C≤6))-heterocycloalkyl_((C≤12)),            —C(O)-alkyl-amino_((C≤12)), —C(O)-dialkylamino_((C≤12)),            —C(O)—N-heterocycloalkyl_((C≤12)), wherein:            -   R₁₁ is hydrogen, alkyl_((C≤6)), or substituted                alkyl_((C≤6));    -   wherein the final degradable diacyl in the chain is attached to        a terminating group;    -   n is 0, 1, 2, 3, 4, 5, or 6;        or a pharmaceutically acceptable salt thereof. In some        embodiments, the dendrimer has the formula:

Core-(Repeating Unit)_(n)-Terminating Group  (I)

-   wherein the core is linked to the repeating unit by removing one or    more hydrogen atoms from the core and replacing the atom with the    repeating unit and wherein:    -   the core has the formula:

-   -   wherein:        -   X₂ is N(R₅)_(y);            -   R₅ is hydrogen or alkyl_((C≤8)), or substituted                alkyl_((C≤18)); and            -   y is 0, 1, or 2, provided that the sum of y and z is 3;        -   R₂ is amino, hydroxy, or mercapto, or alkylamino_((C≤12)),            dialkylamino_((C≤12)), or a substituted version of either of            these groups;        -   b is 1, 2, 3, 4, 5, or 6; and        -   z is 1, 2, 3; provided that the sum of z and y is 3;    -   wherein the repeating unit comprises a degradable diacyl and a        linker;        -   the degradable diacyl group has the formula:

-   -   -   wherein:            -   A₁ and A₂ are each independently —O— or —NR_(a)—,                wherein:                -   R_(a) is hydrogen, alkyl_((C≤6)), or substituted                    alkyl_((C≤6));            -   Y₃ is alkanediyl_((C≤12)), alkenediyl_((C≤12)),                arenediyl_((C≤12)), or a substituted version of any of                these groups; or a group of the formula:

-   -   -   -   wherein:                -   X₃ and X₄ are alkanediyl_((C≤12)),                    alkenediyl_((C≤12)), arenediyl_((C≤12)), or a                    substituted version of any of these groups;                -   Y₅ is a covalent bond, alkanediyl_((C≤12)),                    alkenediyl_((C≤12)), arenediyl_((C≤12)), or a                    substituted version of any of these groups; and            -   R₉ is alkyl_((C≤8)) or substituted alkyl_((C≤8));

        -   the linker group has the formula:

-   -   -   wherein:            -   Y₁ is alkanediyl_((C≤12)), alkenediyl_((C≤12)),                arenediyl_((C≤12)), or a substituted version of any of                these groups; and

    -   wherein when the repeating unit comprises a linker group, then        the linker group is attached to a degradable diacyl group on        both the nitrogen and the sulfur atoms of the linker group,        wherein the first group in the repeating unit is a degradable        diacyl group, wherein for each linker group, the next group        comprises two degradable diacyl groups attached to the nitrogen        atom of the linker group; and wherein n is the number of linker        groups present in the repeating unit; and

    -   the terminating group, wherein the terminating group has the        formula:

-   -   wherein:        -   Y₄ is alkanediyl_((C≤18)) or an alkanediyl_((C≤18)) wherein            one or more of the hydrogen atoms on the alkanediyl_((C≤18))            has been replaced with —OH, —F, —Cl, —Br, —I, —SH, —OCH₃,            —OCH₂CH₃, —SCH₃, or —OC(O)CH₃;        -   R₁₀ is hydrogen, carboxy, hydroxy, or        -   aryl_((C≤12)), alkylamino_((C≤12)), dialkylamino_((C≤12)),            N-heterocycloalkyl_((C≤12)),            —C(O)N(R₁₁)-alkanediyl_((C≤6))-heterocycloalkyl_((C≤12)),            —C(O)-alkyl-amino_((C≤12)), —C(O)-dialkylamino_((C≤12)),            —C(O)—N-heterocycloalkyl_((C≤12)), wherein:    -   wherein the final degradable diacyl in the chain is attached to        a terminating group;    -   n is 0, 1, 2, 3, 4, 5, or 6;        or a pharmaceutically acceptable salt thereof. In other        embodiments, the dendrimer has the formula:

Core-(Repeating Unit)_(n)-Terminating Group  (I)

-   wherein the core is linked to the repeating unit by removing one or    more hydrogen atoms from the core and replacing the atom with the    repeating unit and wherein:    -   the core has the formula:

-   -   wherein:        -   X₃ is —NR₆—, wherein R₆ is hydrogen, alkyl_((C≤8)), or            substituted alkyl_((C≤8)), —O—, or alkylaminodiyl_((C≤8)),            alkoxydiyl_((C≤8)), arenediyl_((C≤8)),            heteroarenediyl_((C≤8)), heterocycloalkanediyl_((C≤8)), or a            substituted version of any of these groups;        -   R₃ and R₄ are each independently amino, hydroxy, or            mercapto, or alkylamino_((C≤12)), dialkylamino_((C≤12)), or            a substituted version of either of these groups; or a group            of the formula: —(CH₂CH₂N)_(e)(R_(c))R_(d);        -   wherein:            -   e is 1, 2, or 3;            -   R_(c) and R_(d) are each independently hydrogen,                alkyl_((C≤6)), or substituted alkyl_((C≤6));        -   c and d are each independently 1, 2, 3, 4, 5, or 6; and    -   wherein the repeating unit comprises a degradable diacyl and a        linker;        -   the degradable diacyl group has the formula:

-   -   -   wherein:            -   A₁ and A₂ are each independently —O— or —NR_(a)—,                wherein:                -   R_(a) is hydrogen, alkyl_((C≤6)), or substituted                    alkyl_((C≤6));            -   Y₃ is alkanediyl_((C≤12)), alkenediyl_((C≤12)),                arenediyl_((C≤12)), or a substituted version of any of                these groups; or a group of the formula:

-   -   -   -   wherein:                -   X₃ and X₄ are alkanediyl_((C≤12)),                    alkenediyl_((C≤12)), arenediyl_((C≤12)), or a                    substituted version of any of these groups;                -   Y₅ is a covalent bond, alkanediyl_((C≤12)),                    alkenediyl_((C≤12)), arenediyl_((C≤12)), or a                    substituted version of any of these groups; and            -   R₉ is alkyl_((C≤8)) or substituted alkyl_((C≤8));

        -   the linker group has the formula:

-   -   -   wherein:            -   Y₁ is alkanediyl_((C≤12)), alkenediyl_((C≤12)),                arenediyl_((C≤12)), or a substituted version of any of                these groups; and

    -   wherein when the repeating unit comprises a linker group, then        the linker group is attached to a degradable diacyl group on        both the nitrogen and the sulfur atoms of the linker group,        wherein the first group in the repeating unit is a degradable        diacyl group, wherein for each linker group, the next group        comprises two degradable diacyl groups attached to the nitrogen        atom of the linker group; and wherein n is the number of linker        groups present in the repeating unit; and

    -   the terminating group, wherein the terminating group has the        formula:

-   -   wherein:        -   Y₄ is alkanediyl_((C≤18)) or an alkanediyl_((C≤18)) wherein            one or more of the hydrogen atoms on the alkanediyl_((C≤18))            has been replaced with —OH, —F, —Cl, —Br, —I, —SH, —OCH₃,            —OCH₂CH₃, —SCH₃, or —OC(O)CH₃;        -   R₁₀ is hydrogen, carboxy, hydroxy, or        -   aryl_((C≤12)), alkylamino_((C≤12)), dialkylamino_((C≤12)),            N-heterocycloalkyl_((C≤12)),            —C(O)N(R₁₁)-alkanediyl_((C≤6))-heterocycloalkyl_((C≤12)),            —C(O)-alkyl-amino_((C≤12)), —C(O)-dialkylamino_((C≤12)),            —C(O)—N-heterocycloalkyl_((C≤12)), wherein:            -   R₁₁ is hydrogen, alkyl_((C≤6)), or substituted                alkyl_((C≤6));    -   wherein the final degradable diacyl in the chain is attached to        a terminating group;    -   n is 0, 1, 2, 3, 4, 5, or 6;        or a pharmaceutically acceptable salt thereof. In other        embodiments, the dendrimer has the formula:

Core-(Repeating Unit)_(n)-Terminating Group  (I)

-   wherein the core is linked to the repeating unit by removing one or    more hydrogen atoms from the core and replacing the atom with the    repeating unit and wherein:    -   the core is alkylamine_((C≤18)), dialkylamine_((C≤36)),        heterocycloalkane_((C≤12)), or a substituted version of any of        these groups; and    -   wherein the repeating unit comprises a degradable diacyl and a        linker;        -   the degradable diacyl group has the formula:

-   -   -   wherein:            -   A₁ and A₂ are each independently —O— or —NR_(a)—,                wherein:                -   R_(a) is hydrogen, alkyl_((C≤6)), or substituted                    alkyl_((C≤6));            -   Y₃ is alkanediyl_((C≤12)), alkenediyl_((C≤12)),                arenediyl_((C≤12)), or a substituted version of any of                these groups; or a group of the formula:

-   -   -   -   wherein:                -   X₃ and X₄ are alkanediyl_((C≤12)),                    alkenediyl_((C≤12)), arenediyl_((C≤12)), or a                    substituted version of any of these groups;                -   Y₅ is a covalent bond, alkanediyl_((C≤12)),                    alkenediyl_((C≤12)), arenediyl_((C≤12)), or a                    substituted version of any of these groups; and            -   R₉ is alkyl_((C≤8)) or substituted alkyl_((C≤8));

        -   the linker group has the formula:

-   -   -   wherein:            -   Y₁ is alkanediyl_((C≤12)), alkenediyl_((C≤12)),                arenediyl_((C≤12)), or a substituted version of any of                these groups; and

    -   wherein when the repeating unit comprises a linker group, then        the linker group is attached to a degradable diacyl group on        both the nitrogen and the sulfur atoms of the linker group,        wherein the first group in the repeating unit is a degradable        diacyl group, wherein for each linker group, the next group        comprises two degradable diacyl groups attached to the nitrogen        atom of the linker group; and wherein n is the number of linker        groups present in the repeating unit; and

    -   the terminating group, wherein the terminating group has the        formula:

-   -   wherein:        -   Y₄ is alkanediyl_((C≤18)) or an alkanediyl_((C≤18)) wherein            one or more of the hydrogen atoms on the alkanediyl_((C≤18))            has been replaced with —OH, —F, —Cl, —Br, —I, —SH, —OCH₃,            —OCH₂CH₃, —SCH₃, or —OC(O)CH₃;        -   R₁₀ is hydrogen, carboxy, hydroxy, or        -   aryl_((C≤12)), alkylamino_((C≤12)), dialkylamino_((C≤12)),            N-heterocycloalkyl_((C≤12)),            —C(O)N(R₁₁)-alkanediyl_((C≤6))-heterocycloalkyl_((C≤12)),            —C(O)-alkyl-amino_((C≤12)), —C(O)-dialkylamino_((C≤12)),            —C(O)—N-heterocycloalkyl_((C≤12)), wherein:            -   R₁₁ is hydrogen, alkyl_((C≤6)), or substituted                alkyl_((C≤6));    -   wherein the final degradable diacyl in the chain is attached to        a terminating group;    -   n is 0, 1, 2, 3, 4, 5, or 6;        or a pharmaceutically acceptable salt thereof. In some        embodiments, the terminating group is further defined by the        formula:

wherein:

-   -   Y₄ is alkanediyl_((C≤18)) or an alkanediyl_((C≤18)) wherein one        or more of the hydrogen atoms has been replaced with —OH, —F,        —Cl, —Br, —I, —SH, —OCH₃, —OCH₂CH₃, —SCH₃, or —OC(O)CH₃; and R₁₀        is hydrogen.        In other embodiments, the terminating group is further defined        by the formula:

wherein:

-   -   Y₄ is alkanediyl_((C≤18)); and R₁₀ is hydrogen.        In some embodiments, Y₄ is alkanediyl_((C4-18)). In other        embodiments, the terminating group is further defined by the        formula:

wherein:

-   -   Y₄ is alkanediyl_((C≤18)) or an alkanediyl_((C≤18)) wherein one        or more of the hydrogen atoms has been replaced with —OH, —F,        —Cl, —Br, —I, —SH, —OCH₃, —OCH₂CH₃, —SCH₃, or —OC(O)CH₃; R₁₀ is        alkylamino_((C≤12)), dialkylamino_((C≤12)),        N-heterocycloalkyl_((C≤12)).        In some embodiments, the terminating group is further defined by        the formula:

wherein:

-   -   Y₄ is alkanediyl_((C≤18)) or an alkanediyl_((C≤18)) wherein one        or more of the hydrogen atoms has been replaced with —OH, —F,        —Cl, —Br, —I, —SH, —OCH₃, —OCH₂CH₃, —SCH₃, or —OC(O)CH₃; R₁₀ is        hydroxy.        In some embodiments, the core is further defined by the formula:

wherein:

-   -   X₁ is alkylamino_((C≤12)), dialkylamino_((C≤12)),        heterocycloalkyl_((C≤12)), heteroaryl_((C≤12)), or a substituted        version thereof;    -   R₁ is amino, hydroxy, or mercapto, or alkylamino_((C≤12)),        dialkylamino_((C≤12)), or a substituted version of either of        these groups; and    -   a is 1, 2, 3, 4, 5, or 6;

In some embodiments, X₁ is alkylamino_((C≤12)) or substitutedalkylamino_((C≤12)). In some embodiments, X₁ is ethylamino. In otherembodiments, X₁ is dialkylamino_((C≤12)) or substituteddialkylamino_((C≤12)). In some embodiments, X₁ is dimethylamino. Inother embodiments, X₁ is heterocycloalkyl_((C≤12)) or substitutedheterocycloalkyl_((C≤12)). In some embodiments, X₁ is 4-piperidinyl,N-piperidinyl, N-morpholinyl, N-pyrrolidinyl, 2-pyrrolidinyl,N-piperazinyl, or N-4-methylpiperadizinyl. In other embodiments, X₁ isheteroaryl_((C≤12)) or substituted heteroaryl_((C≤12)). In someembodiments, X₁ is 2-pyridinyl or N-imidazolyl. In some embodiments, R₁is hydroxy. In other embodiments, R₁ is amino. In other embodiments, R₁is alkylamino_((C≤12)) or substituted alkylamino_((C≤12)). In someembodiments, R₁ is alkylamino_((C≤12)). In some embodiments, R₁ ismethylamino or ethylamino. In some embodiments, a is 1, 2, 3, or 4. Insome embodiments, a is 2 or 3. In some embodiments, a is 2. In otherembodiments, a is 3. In some embodiments, the core is further defined asa compound of the formula:

In some embodiments, the core is further defined as:

In other embodiments, the core is further defined by the formula:

wherein:

-   -   X₂ is N(R₅)_(y);        -   R₅ is hydrogen or alkyl_((C≤8)), or substituted            alkyl_((C≤18)); and        -   y is 0, 1, or 2, provided that the sum of y and z is 3;    -   R₂ is amino, hydroxy, or mercapto, or alkylamino_((C≤12)),        dialkylamino_((C≤12)), or a substituted version of either of        these groups;    -   b is 1, 2, 3, 4, 5, or 6; and    -   z is 1, 2, 3; provided that the sum of z and y is 3.

In some embodiments, X₂ is N. In other embodiments, X₂ is NR₅, whereinR₅ is hydrogen or alkyl_((C≤8)). In some embodiments, R₅ is hydrogen. Inother embodiments, R₅ is methyl. In some embodiments, z is 3. In otherembodiments, z is 2. In some embodiments, R₂ is hydroxy. In otherembodiments, R₂ is amino. In other embodiments, R₂ isalkylamino_((C≤12)) or substituted alkylamino_((C≤12)). In someembodiments, R₂ is alkylamino_((C≤12)). In some embodiments, R₂ ismethylamino. In other embodiments, R₂ is dialkylamino_((C≤12)) orsubstituted dialkylamino_((C≤12)). In some embodiments, R₂ isdialkylamino_((C≤12)). In some embodiments, R₂ is dimethylamino. In someembodiments, b is 1, 2, 3, or 4. In some embodiments, b is 2 or 3. Insome embodiments, b is 2. In other embodiments, b is 3. In someembodiments, the core is further defined as:

In some embodiments, the core is further defined as:

In other embodiments, the core is further defined as:

wherein:

-   -   X₃ is —NR₆—, wherein R₆ is hydrogen, alkyl_((C≤8)), or        substituted alkyl_((C≤8)), —O—, or alkylaminodiyl_((C≤8)),        alkoxydiyl_((C≤8)), arenediyl_((C≤8)), heteroarenediyl_((C≤8)),        heterocycloalkanediyl_((C≤8)), or a substituted version of any        of these groups;    -   R₃ and R₄ are each independently amino, hydroxy, or mercapto, or        alkylamino_((C≤12)), dialkylamino_((C≤12)), or a substituted        version of either of these groups; or a group of the formula:        —(CH₂CH₂N)_(e)(R_(c))R_(d);        -   wherein:            -   e is 1, 2, or 3;            -   R_(c) and R_(d) are each independently hydrogen,                alkyl_((C≤6)), or substituted alkyl_((C≤6));    -   c and d are each independently 1, 2, 3, 4, 5, or 6.

In some embodiments, X₃ is —O—. In other embodiments, X₃ is —NR₆—,wherein R₆ is hydrogen, alkyl_((C≤8)), or substituted alkyl_((C≤8)). Insome embodiments, X₃ is —NH— or —NCH₃—. In other embodiments, X₃ isalkylaminodiyl_((C≤8)) or substituted alkylaminodiyl_((C≤8)). In someembodiments, X₃ is —NHCH₂CH₂NH— or —NHCH₂CH₂NHCH₂CH₂NH—. In otherembodiments, X₃ is alkoxydiyl_((C≤8)) or substituted alkoxydiyl_((C≤8)).In some embodiments, X₃ is —OCH₂CH₂O—. In other embodiments, X₃ isarenediyl_((C≤8)) or substituted arenediyl_((C≤8)). In some embodiments,X₃ is benzenediyl. In other embodiments, X₃ isheterocycloalkanediyl_((C≤8)) or substitutedheterocycloalkanediyl_((C≤8)). In some embodiments, X₃ isN,N′-piperazindiyl.

In some embodiments, R₃ is amino. In other embodiments, R₃ is hydroxy.In other embodiments, R₃ is alkylamino_((C≤12)) or substitutedalkylamino_((C≤12)). In some embodiments, R₃ is alkylamino_((C≤12)). Insome embodiments, R₃ is methylamino. In other embodiments, R₃ isdialkylamino_((C≤12)) or substituted dialkylamino_((C≤12)). In someembodiments, R₃ is dialkylamino_((C≤12)). In some embodiments, R₃ isdimethylamino.

In some embodiments, R₄ is amino. In other embodiments, R₄ is hydroxy.In other embodiments, R₄ is alkylamino_((C≤12)) or substitutedalkylamino_((C≤12)). In some embodiments, R₄ is alkylamino_((C≤12)). Insome embodiments, R₄ is methylamino. In other embodiments, R₄ isdialkylamino_((C≤12)) or substituted dialkylamino_((C≤12)). In someembodiments, R₄ is dialkylamino_((C≤12)). In some embodiments, R₄ isdimethylamino. In other embodiments, R₄ is —(CH₂CH₂N)_(e)(R_(c))R_(d):wherein: e is 1, 2, or 3; and R_(c) and R_(d) are each independentlyhydrogen, alkyl_((C≤6)), or substituted alkyl_((C≤6)). In someembodiments, e is 1 or 2. In some embodiments, e is 1. In someembodiments, R_(c) is hydrogen. In some embodiments, R_(d) is hydrogen.

In some embodiments, c is 1, 2, 3, or 4. In some embodiments, c is 2 or3. In some embodiments, c is 2. In other embodiments, c is 3. In someembodiments, d is 1, 2, 3, or 4. In some embodiments, d is 2 or 3. Insome embodiments, d is 2. In other embodiments, d is 3. In someembodiments, the core is further defined as:

In some embodiments, the core is further defined as:

In other embodiments, the core is alkylamine_((C≤18)),dialkylamine_((C≤36)), heterocycloalkane_((C≤12)), or a substitutedversion of any of these groups. In some embodiments, the core is analkylamine_((C≤18)) or substituted alkylamine_((C≤18)). In someembodiments, the core is octylamine, decylamine, dodecylamine,tetradecylamine, hexadecylamine, and octadecylamine. In otherembodiments, the core is an dialkylamine_((C≤36)) or substituteddialkylamine_((C≤36)). In some embodiments, the core is N-methyl,N-dodecylamine, dioctylamine, or didecylamine. In other embodiments, thecore is heterocycloalkane_((C≤12)) or substitutedheterocycloalkane_((C≤12)). In some embodiments, the core is4-N-methylpiperazinyl. In some embodiments, Y₁ is alkanediyl_((C≤8)) orsubstituted alkanediyl_((C≤8)). In some embodiments, Y₁ isalkanediyl_((C≤8)). In some embodiments, Y₁ is —CH₂CH₂—. In someembodiments, Y₃ is alkanediyl_((C≤8)) or substituted alkanediyl_((C≤8)).In some embodiments, Y₃ is alkanediyl_((C≤8)). In some embodiments, Y₃is —CH₂CH₂—. In other embodiments, Y₃ is:

wherein:

-   -   X₃ and X₄ are alkanediyl_((C≤12)), alkenediyl_((C≤12)),        arenediyl_((C≤12)), or a substituted version of any of these        groups;    -   Y₅ is a covalent bond, alkanediyl_((C≤12)), alkenediyl_((C≤12)),        arenediyl_((C≤12)), or a substituted version of any of these        groups.

In some embodiments, X₃ is alkanediyl_((C≤12)) or substitutedalkanediyl_((C≤12)). In some embodiments, X₃ is —CH₂CH₂—. In someembodiments, X₄ is alkanediyl_((C≤12)) or substitutedalkanediyl_((C≤12)). In some embodiments, X₄ is —CH₂CH₂—. In someembodiments, Y₅ is a covalent bond. In some embodiments, Y₃ is:

wherein:

-   -   X₃ and X₄ are alkanediyl_((C≤12)), alkenediyl_((C≤12)),        arenediyl_((C≤12)), or a substituted version of any of these        groups;    -   Y₅ is a covalent bond, alkanediyl_((C≤12)), alkenediyl_((C≤12)),        arenediyl_((C≤12)), or a substituted version of any of these        groups.

In some embodiments, X₃ is alkanediyl_((C≤12)) or substitutedalkanediyl_((C≤12)). In some embodiments, X₃ is —CH₂CH₂—. In someembodiments, X₄ is alkanediyl_((C≤12)) or substitutedalkanediyl_((C≤12)). In some embodiments, X₄ is —CH₂CH₂—. In someembodiments, Y₅ is a covalent bond. In some embodiments, Y₅ is —CH₂— or—C(CH₃)₂—. In some embodiments, A₁ is —O—. In other embodiments, A₁ is—NR_(a)—. In some embodiments, R_(a) is hydrogen. In some embodiments,A₂ is —O—. In other embodiments, A₂ is —NR_(a)—. In some embodiments,R_(a) is hydrogen. In some embodiments, R₉ is alkyl_((C≤8)). In someembodiments, R₉ is methyl. In some embodiments, n is 0, 1, 2, 3 or 4. Insome embodiments, n is 0, 1, 2, or 3. In some embodiments, n is 0. Inother embodiments, n is 1. In other embodiments, n is 2. In otherembodiments, n is 3.

In yet another aspect, the present disclosure provides compositionscomprising:

(a) a dendrimer described herein; and

(b) a nucleic acid.

In some embodiments, the nucleic acid is a short interfering RNA (e.g.small interfering RNA) (siRNA), a microRNA (miRNA), a pri-miRNA, amessenger RNA (mRNA), a cluster regularly interspaced short palindromicrepeats (CRISPR) related nucleic acid, a single guide RNA (sgRNA), aCRISPR-RNA (crRNA), a trans-activating crRNA (tracrRNA), a plasmid DNA(pDNA), a transfer RNA (tRNA), an antisense oligonucleotide (ASO), aguide RNA, a double stranded DNA (dsDNA), a single stranded DNA (ssDNA),a single stranded RNA (ssRNA), and a double stranded RNA (dsRNA). Insome embodiments, the nucleic acid is a siRNA, a tRNA, or a nucleic acidwhich may be used in a CRISPR process. The nucleic acid may be a siRNA.In other embodiments, the nucleic acid which may be used in a CRISPRprocess such as a cluster regularly interspaced short palindromicrepeats (CRISPR) related nucleic acid, a single guide RNA (sgRNA), aCRISPR-RNA (crRNA), or a trans-activating crRNA (tracrRNA). In someembodiments, the nucleic acid is a siRNA against Factor VII comprisingthe sequence:

-   -   5′-GGAucAucucAAGucuuAc[dT][dT]-3′ (SEQ ID NO: 1); or    -   3′-GuAAGAcuuGAGAuGAucc[dT][dT]-5′ (SEQ ID NO: 2).

In other embodiments, the nucleic acid is a miRNA. In other embodiments,the nucleic acid is a mRNA. In other embodiments, the nucleic acid is atRNA. In other embodiments, the nucleic acid is a guide RNA. In someembodiments, the guide RNA is used in CRISPR processes. In otherembodiments, the nucleic acid is a pDNA.

In some embodiments, the dendrimer and the nucleic acid are present in aweight ratio from about 100:1 to about 1:5. In some embodiments, theweight ratio of dendrimer to nucleic acid is from about 50:1 to about2:1. In some embodiments, the weight ratio of dendrimer to nucleic acidis 25:1. In other embodiments, the weight ratio of dendrimer to nucleicacid is 7:1. In some embodiments, the composition further comprises oneor more helper lipids. In some embodiments, the helper lipid is selectedfrom a steroid, a steroid derivative, a PEG lipid, or a phospholipid. Insome embodiments, the helper lipid is a steroid or steroid derivative.In some embodiments, the steroid is cholesterol. In some embodiments,the steroid or steroid derivative and the dendrimer are present in amolar ratio from about 10:1 to about 1:20. In some embodiments, themolar ratio of the steroid or steroid derivative and dendrimer is fromabout 1:1 to about 1:10. In some embodiments, the molar ratio of thesteroid or steroid derivative and dendrimer is about 38:50. In someembodiments, the molar ratio of the steroid or steroid derivative anddendrimer is about 1:5.

In other embodiments, the helper lipid is a PEG lipid. In someembodiments, the PEG lipid is a PEGylated diacylglycerol such as acompound of the formula:

-   -   wherein:        -   R₁₂ and R₁₃ are each independently alkyl_((C≤24)),            alkenyl_((C≤24)), or a substituted version of either of            these groups;        -   R_(e) is hydrogen, alkyl_((C≤8)), or substituted            alkyl_((C≤8)); and x is 1-250.

In some embodiments, the PEG lipid is dimyristoyl-sn-glycerol or acompound of the formula:

wherein:

-   -   n₁ is 5-250; and    -   n₂ and n₃ are each independently 2-25.

In some embodiments, the PEG lipid and the dendrimer are present in amolar ratio from about 1:1 to about 1:250. In some embodiments, themolar ratio of the PEG lipid and the dendrimer is from about 1:10 toabout 1:125. In some embodiments, the molar ratio of the PEG lipid andthe dendrimer is from about 1:20 to about 1:50.

In other embodiments, the helper lipid is a phospholipid. In someembodiments, the phospholipid is1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC). In other embodiments,the phospholipid is 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine(DOPE). In some embodiments, the phospholipid and the dendrimer arepresent in a molar ratio from about 10:1 to about 1:20. In someembodiments, the molar ratio of the phospholipid and dendrimer is fromabout 1:1 to about 1:10. In some embodiments, the molar ratio of thephospholipid and dendrimer is about 4:5. In some embodiments, the molarratio of the phospholipid and dendrimer is about 1:5. In someembodiments, the composition consists essentially of the dendrimer, thenucleic acid, and one or more helper lipids.

In still yet another aspect, the present disclosure providespharmaceutical composition comprising:

-   -   (a) a composition or dendrimer described herein; and    -   (b) a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical acceptable carrier is a solventor solution. In some embodiments, the pharmaceutical composition isformulated for administration: orally, intraadiposally, intraarterially,intraarticularly, intracranially, intradermally, intralesionally,intramuscularly, intranasally, intraocularly, intrapericardially,intraperitoneally, intrapleurally, intraprostatically, intrarectally,intrathecally, intratracheally, intratumorally, intraumbilically,intravaginally, intravenously, intravesicularlly, intravitreally,liposomally, locally, mucosally, parenterally, rectally,subconjunctival, subcutaneously, sublingually, topically, transbuccally,transdermally, vaginally, in cremes, in lipid compositions, via acatheter, via a lavage, via continuous infusion, via infusion, viainhalation, via injection, via local delivery, or via localizedperfusion. In some embodiments, the pharmaceutical composition isformulated for intravenous or intraarterial injection. In someembodiments, the pharmaceutical composition is formulated as a unitdose.

In yet another aspect, the present disclosure provides methods ofmodulating the expression of a gene comprising delivering a nucleic acidto a cell, the methods comprising contacting the cell with a compositionor a pharmaceutical composition described herein under conditionssufficient to cause uptake of the nucleic acid into the cell. In someembodiments, the cell is contacted in vitro. In other embodiments, thecell is contacted in vivo. In other embodiments, the cell is contactedex vivo. In some embodiments, the modulation of the gene expression issufficient to treat a disease or disorder. In some embodiments, thedisease or disorder is cancer. In some embodiments, the disease ordisorder is liver cancer. In some embodiments, the disease or disorderis hepatocellular carcinoma.

In still yet another aspect, the present disclosure provides methods oftreating a disease or disorder in a patient comprising administering tothe patient in need thereof a pharmaceutically effective amount of acomposition or a pharmaceutical composition described herein. In someembodiments, the disease or disorder is cancer. In some embodiments, thedisease or disorder is liver cancer. In some embodiments, the disease ordisorder is hepatocellular carcinoma. In some embodiments, the methodsfurther comprise administering one or more additional cancer therapiesto the patient. In some embodiments, the cancer therapy is achemotherapeutic compound, surgery, radiation therapy, or immunotherapy.In some embodiments, the compositions or pharmaceutical compositions areadministered to the patient once. In other embodiments, the compositionsor pharmaceutical compositions are administered to the patient two ormore times. In some embodiments, the patient is a mammal such as ahuman.

In still yet another aspects, the present disclosure provides dendrimersof the formula:

Core-(Repeating Unit)_(n)-Terminating Group  (I)

-   wherein the core is linked to the repeating unit by removing one or    more hydrogen atoms from the core and replacing the atom with the    repeating unit and wherein:    -   the core has the formula:

-   -   wherein:        -   X₃ is —NR₆—, wherein R₆ is hydrogen, alkyl_((C≤8)), or            substituted alkyl_((C≤8)), —O—, or alkylaminodiyl_((C≤8)),            alkoxydiyl_((C≤8)), arenediyl_((C≤8)),            heteroarenediyl_((C≤8)), heterocycloalkenediyl_((C≤8)), or a            substituted version of any of these groups;        -   R₃ and R₄ are each independently amino, hydroxy, or            mercapto, or alkylamino_((C≤12)), dialkylamino_((C≤12)), or            a substituted version of either of these groups;        -   c and d are each independently 1, 2, 3, 4, 5, or 6; or    -   wherein the repeating unit comprises a degradable diacyl and a        linker;        -   the degradable diacyl group has the formula:

-   -   -   wherein:            -   A₁ and A₂ are each independently —O— or —NR_(a)—,                wherein:                -   R_(a) is hydrogen, alkyl_((C≤6)), or substituted                    alkyl_((C≤6));            -   Y₃ is alkanediyl_((C≤12)), alkenediyl_((C≤12)),                arenediyl_((C≤12)), or a substituted version of any of                these groups; and            -   R₉ is alkyl_((C≤8)) or substituted alkyl_((C≤8));        -   the linker group has the formula:

-   -   -   wherein:            -   Y₁ is alkanediyl_((C≤12)), alkenediyl_((C≤12)),                arenediyl_((C≤12)), or a substituted version of any of                these groups; and

    -   wherein when the repeating unit comprises a linker group, then        the linker group is attached to a degradable diacyl group on        both the nitrogen and the sulfur atoms of the linker group,        wherein the first group in the repeating unit is a degradable        diacyl group, wherein for each linker group, the next group        comprises two degradable diacyl groups attached to the nitrogen        atom of the linker group; and wherein n is the number of linker        groups present in the repeating unit; and

    -   the terminating group has the formula:

-   -   wherein:        -   Y₄ is alkanediyl_((C≤18)); and        -   R₁₀ is hydrogen;    -   wherein the final degradable diacyl in the chain is attached to        a terminating group;    -   n is 0, 1, 2, 3, 4, 5, or 6;        or a pharmaceutically acceptable salt thereof.

The terms “comprise” (and any form of comprise, such as “comprises” and“comprising”), “have” (and any form of have, such as “has” and“having”), “contain” (and any form of contain, such as “contains” and“containing”), and “include” (and any form of include, such as“includes” and “including”) are open-ended linking verbs. As a result, amethod, composition, kit, or system that “comprises,” “has,” “contains,”or “includes” one or more recited steps or elements possesses thoserecited steps or elements, but is not limited to possessing only thosesteps or elements; it may possess (i.e., cover) elements or steps thatare not recited. Likewise, an element of a method, composition, kit, orsystem that “comprises,” “has,” “contains,” or “includes” one or morerecited features possesses those features, but is not limited topossessing only those features; it may possess features that are notrecited.

Any embodiment of any of the present methods, composition, kit, andsystems may consist of or consist essentially of—rather thancomprise/include/contain/have—the described steps and/or features. Thus,in any of the claims, the term “consisting of” or “consistingessentially of” may be substituted for any of the open-ended linkingverbs recited above, in order to change the scope of a given claim fromwhat it would otherwise be using the open-ended linking verb.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

Other objects, features and advantages of the present disclosure willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating specific embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.Note that simply because a particular compound is ascribed to oneparticular generic formula doesn't mean that it cannot also belong toanother generic formula.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1A-1D show a combination of high potency to tumor cells and lowtoxicity to normal cells is required for small RNA therapies withinvulnerable liver cancers. A modular strategy for diversifying thechemical functionality and size of biocompatible, ester-based dendrimersallowed discovery of dendrimers that balance low toxicity and high invivo small RNA delivery potency. Orthogonal reactions accelerated thesynthesis of 1,512 G1 modular degradable dendrimers, thereby increasingthe number, size, and chemical diversity of molecular structures.Inclusion of degradable ester bonds at each step promoted low toxicity.(FIG. 1A) Small RNAs are too large and anionic to enter cells on theirown. To efficiently utilize the RNAi machinery, delivery carriers mustchaperone small RNAs through numerous extracellular and intracellularbarriers. A modular design which would enable fine tuning of functionalgroup identity and placement within dendrimer architectures wasenvisioned. (FIG. 1B) The library was established via sequentialorthogonal reactions. First, amines with a series of initial branchingcenters (IBCs) reacted quantitatively and selectively with the lesssterically hindered acrylate groups of AEMA containing two degradableester groups. The products then underwent DMPP-catalyzed reaction withvarious thiols. (FIG. 1C) To identify degradable dendrimers withoptimized topological structures to mediate small RNAs to overcome themultiple extracellular and intracellular delivery barriers, the libraryis divided into four zones: core binding—periphery stabilization (zoneI), core binding—periphery binding (zone II), corestabilization—periphery stabilization (zone III), and corestabilization—periphery binding (zone IV). (FIG. 1D) Dendrimers areindependently modulated with chemically diverse amines and thiols forcores and peripheries. Selected amines are divided into two categories:ionizable amines for RNA binding that will generate one to six branchedspecies are labeled 1A-6A and hydrophobic amines for NP stabilizationare labeled 1H-2H. These amines are expected to increase potency withhigher dendrimer generation. Hydrophobic alkyl amines for NPstabilization are labeled SC1-SC19 based on the hydrocarbon length.Alcohol and carboxylic acid terminated thiols (SO1-SO9) andamine-functionalized thiols (SN1-SN11) are included in the librarydesign to increase chemical diversity. G2-G4 higher generationdendrimers with multiple branches were also synthesized using generationexpansion reactions (see FIG. 10B and FIG. 11).

FIG. 2 shows high aza-Michael addition selectivity oftris(2-aminoethyl)amine 6A3 with 2-(acryloyloxy)ethyl methacrylate(AEMA) in the presence of 5 mol % of butylated hydroxyltoluene (BHT) at50° C. Without addition of tris(2-aminoethyl)amine, AEMA alone isunreacted after 24 hours and its conversion is 0%. After addingtris(2-aminoethyl)amine, AEMA conversion is around 82% after 2 hours and98% after 24 hours to generate a first-generation dendrimer with sixbranches, 6A3-G1. Note that excess EAMA (6×0.05 eq.) is added with aimto easily monitor the reaction by ¹H NMR tracking of EAMA. If EAMAconversion is complete, there should still be 5% signal of H_(a) andH_(b) remaining.

FIG. 3 shows high aza-Michael addition selectivity of long alkyl chaintetradecylamine 2H4 with 2-(acryloyloxy)ethyl methacrylate (AEMA) in thepresence of 5 mol % of butylated hydroxyltoluene (BHT) at 50° C. AEMAalone is unreacted after 24 hours and its conversion is 0%. After addingtetradecylamine, AEMA conversion is around 97% after 24 hours togenerate a first-generation dendrimer with long alkyl chain core 2H4-G1.Note that excess EAMA (2×0.05 eq.) is added with the purpose to easilymonitor the reaction by ¹H NMR tracking of EAMA. If EAMA conversion iscomplete, there should still be 5% signal of H_(a) and H_(b) remaining.

FIG. 4 shows sulfa-Michael addition of 6A3-G1 (125 mM) with2-(butylamino)ethanethiol (6×1.2 eq.) or 1-tetradecanethiol (6×1.2 eq.)at 60° C. in 400 μL DMSO-D6 for 48 hours. Without addition of thiolcompounds, 6A3-G1 remains the same at 60° C. in DMSO-D6 for 48 hours.With addition of (5 mol %) dimethylphenylphosphine (DMPP) as a catalyst,6A3-G1 reacts with 1-tetradecanethiol at 100% conversion yield at 60° C.in DMSO-D6 within 48 hours while the conversion yield of 6A3-G1 is only57% without DMPP. The conversion yield of 6A3-G1 with2-(butylamino)ethanethiol is nearly quantitative with or without theaddition of DMPP, probably because the amine group in2-(butylamino)ethanethiol may act as a catalyst. Note that excess thiol(6×0.2 eq.) is added because 6A3-G1 contains (6×0.05 eq.) EAMA whichconsumes thiol reactant (6×0.1 eq.) with its two double bonds.

FIG. 5 shows sulfa-Michael addition of 2H4-G1 (125 mM) with2-(butylamino)ethanethiol (2×1.2 eq.) or 1-tetradecanethiol (2×1.2 eq.)at 60° C. in DMSO-D6 for 48 hours. Without addition of thiol compounds,2H4-G1 remains the same at 60° C. in DMSO-D6 for 48 hours. With additionof (5 mol %) dimethylphenylphosphine (DMPP) as a catalyst, 2H4-G1 reactswith 1-tetradecanethiol at 100% conversion yield at 60° C. in DMSO-D6within 48 hours while the conversion yield of 2H4-G1 is only 51% withoutDMPP. The conversion yield of 2H4-G1 with 2-(butylamino)ethanethiol isquantitative with or without the addition of DMPP probably because theamine group in 2-(butylamino)ethanethiol may act as a catalyst. Notethat excess thiol (2×0.2 eq.) is added because 2H4-G1 contains (2×0.05eq.) EAMA which consumes thiol reactant (2×0.1 eq.) with its two doublebonds.

FIGS. 6A & 6B show the library of 1,512 first-generation degradabledendrimers was established with high efficiency. (FIG. 6A) The differentamines C with various initial branching centers (IBCs) reacted with2-(acryloyloxy)ethyl methacrylate (AEMA) at exact 1:1 feed equivalencein the presence of 5 mol % of butylated hydroxyltoluene (BHT) at 50° C.for 24 hours. Conversion yield of all 42 reactions is nearlyquantitative by ¹H NMR. (FIG. 6B) Each of 42 C-L-G1s reacted with eachof 36 thiols (P) in 66 μL DMSO with 5% DMPP at small scale (˜20 mg onaverage). The thiol concentration is 750 mM and the concentration of1An&1Hn, 2An&2Hn, 3An, 4An, 5An, and 6An is 750 mM, 275 mM, 250 mM,187.5 mM, 150 mM and 125 mM, respectively. Without addition of any thiolcompounds, all 42 C-L-G1s remained stable at 60° C. for 48 hours. Eachreaction of all 42 with SC4, SN8, and SO9 has nearly quantitativeconversion (measured by ¹H NMR).

FIGS. 7A-7C show in vitro siRNA delivery screening of 1,512 G1DDsenabled discovery of dendrimers that can overcome intracellular barriersand established structure-activity relationships (FIG. 7A). (FIG. 7B) Aheat map of luciferase silencing in HeLa-Luc cells after treatment withdendrimer nanoparticles (33 nM siLuc, n=3) illustrates zone activityrelationships. Luciferase activity and cell viability were measured toidentify dendrimers that balance high delivery potency with low toxicity(see additional data in FIG. 8). (FIG. 7C) Analysis of nanoparticlegroups that enabled more than 50% silencing identified dendrimers withoptimized topological structures to overcome the intracellular deliverybarriers. The daughter zone was further analyzed if its hit rate washigher than that of its parental zone under a series of criteria. Thehit rate of the parental zone is marked in orange, and higher or lowerhit rate of its daughter group is marked in green or blue, respectively.˜6% of whole library enabled >50% gene silencing. The corebinding—periphery stabilization zone I had a 10% hit rate. Within zoneI, the subzone with SC branches yielded 15%, while subzone with SObranches had only a 1% hit rate. In the subzone with SC branches,dendrimers with three to six branches, SC5-8 branches, or SC9-12branches had a much higher chance to efficiently mediate siRNA delivery.

FIG. 8 shows cell viability after addition of 1,512 first-generationdegradable dendrimer (G1DD) NPs containing siLuc (33 nM siRNA, value isaverage of n=3). G1DDs were formulated into nanoparticles containing thefirefly luciferase-targeting siRNA (siLuc) with weight ratio of 12.5:1(G1DD:siRNA) and the helper lipids cholesterol,1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and lipid PEG2000 withmolar ratio of 50:38:10:2 (G1DD:cholesterol:DSPC:lipid PEG). Cellviability was measured with ONE-Glo+Tox luciferase reporter and cellviability assay (Promega) by following its protocol. Cell viability wasobtained by normalizing to untreated cells. Untreated control (n=6).Experimental samples (n=3).

FIG. 9A & 9B show intracellular siRNA delivery activity of 1,512first-generation degradable dendrimers (G1DDs). G1DDs were formulatedinto nanoparticles containing firefly luciferase-targeting siRNA (siLuc)with a weight ratio of 12.5:1 (G1DD:siRNA) and the helper lipidscholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and lipidPEG2000 with molar ratio of 50:38:10:2 (G1DD:cholesterol:DSPC:lipidPEG). (FIG. 9A) The heat map of luciferase activity reduction in HeLacell stably expressing firefly luciferases after treatment of G1DDnanoparticles with 33 nM siRNA is divided into zones and regions todescribe the breakdown of the dendritic analysis process (see part FIG.9B). Cell viability and luciferase activity was measured withONE-Glo+Tox Luciferase reporter and cell viability assay (Promega) byfollowing its protocol. Luciferase reduction was obtained by normalizingluciferase activity to the luciferase activity and viability ofuntreated cells. Untreated control (n=6). Experimental samples (n=3).(FIG. 9B) Utilization of a dendrimer-inspired tree analysis process toidentify degradable dendrimers with optimized structures to mediatesiRNA to overcome the intracellular delivery barriers by analyzing theirhit rate with luciferase activity reduction more than 50%. The daughterzone is further analyzed if its hit rate is higher than that of itsparental zone under a series of standards. Hit rate of parental zone iswith the black bar graph, and higher or low hit rate of its daughtergroup is with the blue or red font. ˜6% of whole library induced >50%gene silencing. The core-binding-periphery-stabilization zone (zone I)has 10% hit rate. In zone I, subzone with SC branches has 15% whilesubzone with SO branches 1%. In subzone with SC branches, dendrimerswith three, four, five, or six branches SC5-8 branches or SC9-12branches have much higher chance efficiently to mediate siRNA toovercome the intracellular delivery barriers.

FIGS. 10A-10C show systematic in vivo siRNA delivery screening furtheridentified dendrimers that can also overcome extracellular barriers.Analysis provided SAR to design additional dendrimers with predictedactivity. (FIG. 10A) 26 first-generation degradable dendrimers withdiversified structures were evaluated for Factor VII knockdown in miceat a siRNA dosage of 1 mg/kg (n=3). PBS control (n=3). Data are shown asmean±s.d. (FIG. 10B) Rational design of degradable dendrimers withmultiple branches was accomplished by (I) choosing polyamines withmultiple IBCs and (II) increasing branching via generation expansion.Natural polyamines spermidine 5A5 and spermine 6A4 were utilized. 1A2(one IBC), 2A2 & 2A11 (two IBCs), 3A3 & 3A5 (three IBCs), and 4A1 & 4A3(four IBCs) were chosen to synthesize degradable dendrimers withmultiple branches via generation expansion (see also FIG. 11). (FIG.10C) 24 rationally designed degradable dendrimers via strategies I andII were evaluated for Factor VII knockdown in mice at a siRNA dosage of1 mg/kg (n=3). PBS control (n=3). Data are shown as mean±s.d. Rationallydesigned dendrimers were active at a high hit rate.

FIG. 11 shows synthetic route of 2A2 & 2A11 (two IBCs), 3A3 & 3A5 (threeIBCs), and 4A1 & 4A3 (four IBCs) to prepare degradable dendrimers withmultiple branches via the generation expansion strategy.

FIGS. 12A-12E show in vivo toxicity evaluation of some of the degradabledendrimers (>95% in vivo FVII knockdown) further identifying dendrimersthat could balance high delivery efficacy with low toxicity. Some of thedegradable dendrimer NPs possessed (FIG. 12A) similar size and (FIG.12B) net surface charge after binding control siRNA (siCTR)(nanoparticles are depicted in the graph from left to right based uponthe legend for FIG. 12B). C12-200 lipidoid LNPs provided a challengingcomparison, as they represent the best example of a non-hydrolyzablesystem with comparable in vivo efficacy. (FIG. 12C) Wild type mice (p26)were injected i.v. with some NPs at 4 mg siCTR/kg (100 mg dendrimer/kgor 28 mg control C12-200/kg) (n=3). Body weight change varied among theformulations according to the dendrimer identity, but all NPs werelargely nontoxic in normal, WT mice. (FIG. 12D) Body weight change oftransgenic mice bearing aggressive MYC-driven tumors (p32) afterinjection with 3 mg siCTR/kg (75 mg/kg 5A2-SC8 and 6A3-SC12 or 21 mg/kgC12-200) (n=5). (FIG. 12E) Kaplan-Meier survival curve of transgenicmice injected at days 32, 36, 40, and 44 with 5A2-SC8 and 6A3-SC12nanoparticles at 3 mg siCTR/kg dosage (75 mg dendrimer/kg) (n=5). Intumor-bearing mice (a vulnerable host), toxicity of the carrier wasexaggerated, and only 5A2-SC8 was able to be well tolerated and notaffect survival. Data are shown as mean±s.d. Statistical analysisperformed with (e) Mantel-Cox test; n.s. P>0.05; *P<0.05.

FIGS. 13A & 13B show the aggressive transgenic MYC-driven liver tumormodel was chosen to evaluate the toxicity and potency of modulardegradable dendrimers to deliver miRNA for suppression of tumor growth(Nguyen et al., 2014). (FIG. 13A) Schematic showing the LAP-tTa; TRE-MYCtransgenic mouse model. TRE-MYC is turned ON or OFF by theliver-specific LAP promoter when present with the LAP-tTA transgene inthe absence or presence of doxycycline (Dox). (FIG. 13B) Without anytreatment, liver tumors are visible around p20-26, then the liver isfull of small tumors by p32, and finally tumors grow large and the liversize increases dramatically at p42 to p60.

FIGS. 14A-14C show fluorescence imaging confirms delivery of siRNA intothe tumor cells inside of the liver. (FIG. 14A) Gross anatomy andfluorescence imaging of transgenic mice bearing aggressive liver tumorsat the age of 41 days. Fluorescence imaging shows that 5A2-SC8nanoparticles formulated with Cy5.5-labeled siRNA mediate massive siRNAaccumulation in the whole cancerous liver, with minor accumulation inthe spleen and kidneys 24 hours after i.v. injection of 1 mgCy5.5-siRNA/kg. To further confirm whether 5A2-SC8 NPs can deliver siRNAin vivo into tumor cells, tumor tissues of the liver were collected,embedded in OTC and sectioned for H&E staining and confocal imaging 24hours after i.v. injection. (FIG. 14B) H&E staining confirms that thelivers contain tumors. The same slides of tumor tissues were scannedusing confocal imaging and were captured under three channels: DAPI fornuclei (blue), FITC for phalloidin-stained actin (green), and Cy5.5 forsiRNA (red). (FIG. 14C) Confocal imaging of the same region shows that5A2-SC8 can efficiently deliver siRNA into tumor cells inside of theliver.

FIGS. 15A & 15B show in (FIG. 15A) the biodistribution in normal, wildtype mice and liver tumor-bearing mice of 5A2-SC8 NPs formulated withCy5.5-labeled siRNA and (FIG. 15B) H&E staining images of livers fromtumor-bearing mice. 5A2-SC8 NPs mediate Cy5.5-labeled siRNA accumulationin the whole liver of both normal mice and liver tumor-bearing mice 24hours after i.v. injection of 1 mg siRNA/kg. H&E staining images showsthat the livers of tumor-bearing mice are full of tumors and that theslide used for confocal imaging contains tumor cells. Note that the sizeof the liver increases as the tumors grow (see proportionally equalboxes in FIG. 15A).

FIGS. 16A-16H show modular degradable dendrimers can deliver atherapeutic Let-7g miRNA mimic to a clinically relevant and aggressive,MYC-driven genetic tumor model, resulting in a significant survivalbenefit. (FIG. 16A) 5A2-SC8 NPs enable silencing of FVII protein intransgenic mice bearing MYC-driven liver tumors, as measured in theblood and (FIG. 16B) in harvested liver tissues (single injection, 1mg/kg, p26 mice, 48 hours post-injection) (siCTR on left and siFVII onright). (FIG. 16C) 5A2-SC8 NPs enable delivery of Let-7g to livertissues of transgenic mice bearing MYC-driven liver tumors (singleinjection, 1 mg/kg, p26 mice, 48 hours post-injection). Let-7gexpression was significantly increased, while other Let-7 family memberswere unaffected (siCTR on left and siFVII on right). (FIG. 16D)Transgenic mice bearing MYC-driven liver tumors were given weekly i.v.injections of 1 mg/kg Let-7g, starting on day 26 (which is afterinitiation of tumor development) until day 61. Mice receiving Let-7g hadvisibly smaller abdomens. (FIG. 16E) Abdominal circumference was smallerfor treated mice compared to controls. (FIG. 16F) Representative imagesof livers from Let-7g mimic and control mimic injected mice show reducedtumor burden. (FIG. 16G) Weekly delivery of miRNA mimics inside of5A2-SC8 NPs did not affect normal weight gain, while delivery of miRNAmimics inside of C12-200 LNPs caused weight loss and death. n=5. (FIG.16H) Delivery of Let-7g weekly from 26 to 61 days extended survival. Allcontrol mice receiving no treatment (n=9) and mice receiving 5A2-SC8 NPs(n=5) containing a control untargeted mimic died around 60 days afterbirth. C12-200 LNP injected mice died prematurely (n=7). #C12-200+CTRmimic experiments were halted because all mice receiving theC12-200+Let-7g mimic injections had died (n=7). Delivery of Let-7ginside of 5A2-SC8 NPs provided a pronounced survival benefit. Data areshown as mean±s.d. Statistical analysis performed with (a,b,c,e)two-tailed Student's t-test, or (h) Mantel-Cox test; n.s. P>0.05;*P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

FIGS. 17A-17C show the delivery of siLuc delivery using dendrimernanoparticles formulated with different combinations of cholesterol,phospholipids, and PEG lipids in HeLa-Luc (FIG. 17A), A549-Luc (FIG.17B), and MDA-MB231-Luc (FIG. 17C).

FIGS. 18A & 18B show (FIG. 18A) the comparison of different compositionformulation with DSPC lipids vs. DOPE lipids with PEG-DMG in deliveringsiLuc to HeLa-Luc. FIG. 18B shows the comparison of differentcomposition formulation with DSPC lipids vs. DOPE lipids with PEG-DHD indelivering siLuc to HeLa-Luc.

FIG. 19 shows delivery of a sgRNA delivery using a nanoparticlecomposition containing a dendrimer or Z120 and with and withoutphospholipid DSPC in the nanoparticle formulation.

FIGS. 20A & 20B show percentage encapsulation of the sgRNA (FIG. 20A)and delivery in HeLa-Luc-Cas9 cells (FIG. 20B).

FIGS. 21A & 21B show the viability of IGROV cells to which Luc mRNA hasbeen delivery after 24 hour incubation (FIG. 21A) and 48 hour incubation(FIG. 21B).

FIG. 22 shows the fluorescence microscopy of cells after treatment withmCherry mRNA showing the delivery of the mRNA to those cells.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

In some aspects, the present disclosure provides lipocationic dendrimerswhich may be used as carriers of nucleic acids. In some embodiments, thedendrimers contain one or more groups which undergoes degradation underphysiological conditions. In some embodiments, the dendrimers areformulated into compositions comprising the dendrimers and one or morenucleic acids. These compositions may also further comprise one or morehelper lipids such as cholesterol and/or a phospholipid. Finally, insome aspects, the present disclosure also provides methods of treatingone or more diseases which may be treated with a nucleic acidtherapeutic using the dendrimer compositions.

A. CHEMICAL DEFINITIONS

When used in the context of a chemical group: “hydrogen” means —H;“hydroxy” means —OH; “oxo” means ═O; “carbonyl” means —C(═O)—; “carboxy”means —C(═O)OH (also written as —COOH or —CO₂H); “halo” meansindependently —F, —Cl, —Br or —I; “amino” means —NH₂; “hydroxyamino”means —NHOH; “nitro” means —NO₂; imino means ═NH; “cyano” means —CN;“isocyanate” means —N═C═O; “azido” means —N₃; in a monovalent context“phosphate” means —OP(O)(OH)₂ or a deprotonated form thereof; in adivalent context “phosphate” means —OP(O)(OH)O— or a deprotonated formthereof, “mercapto” means —SH; and “thio” means ═S; “sulfonyl” means—S(O)₂—; “hydroxysulfonyl” means —S(O)₂OH; “sulfonamide” means—S(O)₂NH₂; and “sulfinyl” means —S(O)—.

In the context of chemical formulas, the symbol “—” means a single bond,“═” means a double bond, and “≡” means triple bond. The symbol “

” represents an optional bond, which if present is either single ordouble. The symbol “

” represents a single bond or a double bond. Thus, for example, theformula

includes

And it is understood that no one such ring atom forms part of more thanone double bond. Furthermore, it is noted that the covalent bond symbol“—”, when connecting one or two stereogenic atoms, does not indicate anypreferred stereochemistry. Instead, it covers all stereoisomers as wellas mixtures thereof. The symbol “

”, when drawn perpendicularly across a bond (e.g.

for methyl) indicates a point of attachment of the group. It is notedthat the point of attachment is typically only identified in this mannerfor larger groups in order to assist the reader in unambiguouslyidentifying a point of attachment. The symbol “

” means a single bond where the group attached to the thick end of thewedge is “out of the page.” The symbol “

” means a single bond where the group attached to the thick end of thewedge is “into the page”. The symbol “

” means a single bond where the geometry around a double bond (e.g.,either E or Z) is undefined. Both options, as well as combinationsthereof are therefore intended. Any undefined valency on an atom of astructure shown in this application implicitly represents a hydrogenatom bonded to that atom. A bold dot on a carbon atom indicates that thehydrogen attached to that carbon is oriented out of the plane of thepaper.

When a group “R” is depicted as a “floating group” on a ring system, forexample, in the formula:

then R may replace any hydrogen atom attached to any of the ring atoms,including a depicted, implied, or expressly defined hydrogen, so long asa stable structure is formed. When a group “R” is depicted as a“floating group” on a fused ring system, as for example in the formula:

then R may replace any hydrogen attached to any of the ring atoms ofeither of the fused rings unless specified otherwise. Replaceablehydrogens include depicted hydrogens (e.g., the hydrogen attached to thenitrogen in the formula above), implied hydrogens (e.g., a hydrogen ofthe formula above that is not shown but understood to be present),expressly defined hydrogens, and optional hydrogens whose presencedepends on the identity of a ring atom (e.g., a hydrogen attached togroup X, when X equals —CH—), so long as a stable structure is formed.In the example depicted, R may reside on either the 5-membered or the6-membered ring of the fused ring system. In the formula above, thesubscript letter “y” immediately following the group “R” enclosed inparentheses, represents a numeric variable. Unless specified otherwise,this variable can be 0, 1, 2, or any integer greater than 2, onlylimited by the maximum number of replaceable hydrogen atoms of the ringor ring system.

For the chemical groups and compound classes, the number of carbon atomsin the group or class is as indicated as follows: “Cn” defines the exactnumber (n) of carbon atoms in the group/class. “C≤n” defines the maximumnumber (n) of carbon atoms that can be in the group/class, with theminimum number as small as possible for the group/class in question,e.g., it is understood that the minimum number of carbon atoms in thegroup “alkenyl_((C≤8))” or the class “alkene_((C≤8))” is two. Comparewith “alkoxy_((C≤10))”, which designates alkoxy groups having from 1 to10 carbon atoms. “Cn-n′” defines both the minimum (n) and maximum number(n′) of carbon atoms in the group. Thus, “alkyl_((C2-10))” designatesthose alkyl groups having from 2 to 10 carbon atoms. These carbon numberindicators may precede or follow the chemical groups or class itmodifies and it may or may not be enclosed in parenthesis, withoutsignifying any change in meaning. Thus, the terms “C5 olefin”,“C5-olefin”, “olefin_((C5))”, and “olefin_(C5)” are all synonymous.

The term “saturated” when used to modify a compound or chemical groupmeans the compound or chemical group has no carbon-carbon double and nocarbon-carbon triple bonds, except as noted below. When the term is usedto modify an atom, it means that the atom is not part of any double ortriple bond. In the case of substituted versions of saturated groups,one or more carbon oxygen double bond or a carbon nitrogen double bondmay be present. And when such a bond is present, then carbon-carbondouble bonds that may occur as part of keto-enol tautomerism orimine/enamine tautomerism are not precluded. When the term “saturated”is used to modify a solution of a substance, it means that no more ofthat substance can dissolve in that solution.

The term “aliphatic” when used without the “substituted” modifiersignifies that the compound or chemical group so modified is an acyclicor cyclic, but non-aromatic hydrocarbon compound or group. In aliphaticcompounds/groups, the carbon atoms can be joined together in straightchains, branched chains, or non-aromatic rings (alicyclic). Aliphaticcompounds/groups can be saturated, that is joined by singlecarbon-carbon bonds (alkanes/alkyl), or unsaturated, with one or morecarbon-carbon double bonds (alkenes/alkenyl) or with one or morecarbon-carbon triple bonds (alkynes/alkynyl).

The term “aromatic” when used to modify a compound or a chemical groupatom means the compound or chemical group contains a planar unsaturatedring of atoms that is stabilized by an interaction of the bonds formingthe ring.

The term “alkyl” when used without the “substituted” modifier refers toa monovalent saturated aliphatic group with a carbon atom as the pointof attachment, a linear or branched acyclic structure, and no atomsother than carbon and hydrogen. The groups —CH₃ (Me), —CH₂CH₃ (Et),—CH₂CH₂CH₃ (n-Pr or propyl), —CH(CH₃)₂(i-Pr, ^(i)Pr or isopropyl),—CH₂CH₂CH₂CH₃ (n-Bu), —CH(CH₃)CH₂CH₃ (sec-butyl),—CH₂CH(CH₃)₂(isobutyl), —C(CH₃)₃ (tert-butyl, t-butyl, t-Bu or ^(t)Bu),and —CH₂C(CH₃)₃ (neo-pentyl) are non-limiting examples of alkyl groups.The term “alkanediyl” when used without the “substituted” modifierrefers to a divalent saturated aliphatic group, with one or twosaturated carbon atom(s) as the point(s) of attachment, a linear orbranched acyclic structure, no carbon-carbon double or triple bonds, andno atoms other than carbon and hydrogen. The groups —CH₂— (methylene),—CH₂CH₂—, —CH₂C(CH₃)₂CH₂—, and —CH₂CH₂CH₂— are non-limiting examples ofalkanediyl groups. An “alkane” refers to the class of compounds havingthe formula H—R, wherein R is alkyl as this term is defined above. Whenany of these terms is used with the “substituted” modifier one or morehydrogen atom has been independently replaced by —OH, —F, —Cl, —Br, —I,—NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃,—NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃,—NHC(O)CH₃, —S(O)₂OH, or —S(O)₂NH₂. The following groups arenon-limiting examples of substituted alkyl groups: —CH₂OH, —CH₂Cl, —CF₃,—CH₂CN, —CH₂C(O)OH, —CH₂C(O)OCH₃, —CH₂C(O)NH₂, —CH₂C(O)CH₃, —CH₂OCH₃,—CH₂OC(O)CH₃, —CH₂NH₂, —CH₂N(CH₃)₂, and —CH₂CH₂Cl. The term “haloalkyl”is a subset of substituted alkyl, in which the hydrogen atom replacementis limited to halo (i.e. —F, —Cl, —Br, or —I) such that no other atomsaside from carbon, hydrogen and halogen are present. The group, —CH₂Clis a non-limiting example of a haloalkyl. The term “fluoroalkyl” is asubset of substituted alkyl, in which the hydrogen atom replacement islimited to fluoro such that no other atoms aside from carbon, hydrogenand fluorine are present. The groups —CH₂F, —CF₃, and —CH₂CF₃ arenon-limiting examples of fluoroalkyl groups.

The term “cycloalkyl” when used without the “substituted” modifierrefers to a monovalent saturated aliphatic group with a carbon atom asthe point of attachment, said carbon atom forming part of one or morenon-aromatic ring structures, no carbon-carbon double or triple bonds,and no atoms other than carbon and hydrogen. Non-limiting examplesinclude: —CH(CH₂)₂ (cyclopropyl), cyclobutyl, cyclopentyl, or cyclohexyl(Cy). The term “cycloalkanediyl” when used without the “substituted”modifier refers to a divalent saturated aliphatic group with two carbonatoms as points of attachment, no carbon-carbon double or triple bonds,and no atoms other than carbon and hydrogen. The group

is a non-limiting example of cycloalkanediyl group. A “cycloalkane”refers to the class of compounds having the formula H—R, wherein R iscycloalkyl as this term is defined above. When any of these terms isused with the “substituted” modifier one or more hydrogen atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂.

The term “alkenyl” when used without the “substituted” modifier refersto an monovalent unsaturated aliphatic group with a carbon atom as thepoint of attachment, a linear or branched, acyclic structure, at leastone nonaromatic carbon-carbon double bond, no carbon-carbon triplebonds, and no atoms other than carbon and hydrogen. Non-limitingexamples include: —CH═CH₂ (vinyl), —CH═CHCH₃, —CH═CHCH₂CH₃, —CH₂CH═CH₂(allyl), —CH₂CH═CHCH₃, and —CH═CHCH═CH₂. The term “alkenediyl” when usedwithout the “substituted” modifier refers to a divalent unsaturatedaliphatic group, with two carbon atoms as points of attachment, a linearor branched, a linear or branched acyclic structure, at least onenonaromatic carbon-carbon double bond, no carbon-carbon triple bonds,and no atoms other than carbon and hydrogen. The groups —CH═CH—,—CH═C(CH₃)CH₂—, —CH═CHCH₂—, and —CH₂CH═CHCH₂— are non-limiting examplesof alkenediyl groups. It is noted that while the alkenediyl group isaliphatic, once connected at both ends, this group is not precluded fromforming part of an aromatic structure. The terms “alkene” and “olefin”are synonymous and refer to the class of compounds having the formulaH—R, wherein R is alkenyl as this term is defined above. Similarly theterms “terminal alkene” and “α-olefin” are synonymous and refer to analkene having just one carbon-carbon double bond, wherein that bond ispart of a vinyl group at an end of the molecule. When any of these termsare used with the “substituted” modifier one or more hydrogen atom hasbeen independently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂. The groups —CH═CHF, —CH═CHCl and —CH═CHBr arenon-limiting examples of substituted alkenyl groups.

The term “alkynyl” when used without the “substituted” modifier refersto a monovalent unsaturated aliphatic group with a carbon atom as thepoint of attachment, a linear or branched acyclic structure, at leastone carbon-carbon triple bond, and no atoms other than carbon andhydrogen. As used herein, the term alkynyl does not preclude thepresence of one or more non-aromatic carbon-carbon double bonds. Thegroups —C≡CH, —C≡CCH₃, and —CH₂C≡CCH₃ are non-limiting examples ofalkynyl groups. An “alkyne” refers to the class of compounds having theformula H—R, wherein R is alkynyl. When any of these terms are used withthe “substituted” modifier one or more hydrogen atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂.

The term “aryl” when used without the “substituted” modifier refers to amonovalent unsaturated aromatic group with an aromatic carbon atom asthe point of attachment, said carbon atom forming part of a one or moresix-membered aromatic ring structure, wherein the ring atoms are allcarbon, and wherein the group consists of no atoms other than carbon andhydrogen. If more than one ring is present, the rings may be fused orunfused. As used herein, the term does not preclude the presence of oneor more alkyl or aralkyl groups (carbon number limitation permitting)attached to the first aromatic ring or any additional aromatic ringpresent. Non-limiting examples of aryl groups include phenyl (Ph),methylphenyl, (dimethyl)phenyl, —C₆H₄CH₂CH₃ (ethylphenyl), naphthyl, anda monovalent group derived from biphenyl. The term “arenediyl” when usedwithout the “substituted” modifier refers to a divalent aromatic groupwith two aromatic carbon atoms as points of attachment, said carbonatoms forming part of one or more six-membered aromatic ringstructure(s) wherein the ring atoms are all carbon, and wherein themonovalent group consists of no atoms other than carbon and hydrogen. Asused herein, the term does not preclude the presence of one or morealkyl, aryl or aralkyl groups (carbon number limitation permitting)attached to the first aromatic ring or any additional aromatic ringpresent. If more than one ring is present, the rings may be fused orunfused. Unfused rings may be connected via one or more of thefollowing: a covalent bond, alkanediyl, or alkenediyl groups (carbonnumber limitation permitting). Non-limiting examples of arenediyl groupsinclude:

An “arene” refers to the class of compounds having the formula H—R,wherein R is aryl as that term is defined above. Benzene and toluene arenon-limiting examples of arenes. When any of these terms are used withthe “substituted” modifier one or more hydrogen atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂.

The term “aralkyl” when used without the “substituted” modifier refersto the monovalent group -alkanediyl-aryl, in which the terms alkanediyland aryl are each used in a manner consistent with the definitionsprovided above. Non-limiting examples are: phenylmethyl (benzyl, Bn) and2-phenyl-ethyl. When the term aralkyl is used with the “substituted”modifier one or more hydrogen atom from the alkanediyl and/or the arylgroup has been independently replaced by —OH, —F, —Cl, —Br, —I, —NH₂,—NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃,—NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃,—NHC(O)CH₃, —S(O)₂OH, or —S(O)₂NH₂. Non-limiting examples of substitutedaralkyls are: (3-chlorophenyl)-methyl, and 2-chloro-2-phenyl-eth-1-yl.

The term “heteroaryl” when used without the “substituted” modifierrefers to a monovalent aromatic group with an aromatic carbon atom ornitrogen atom as the point of attachment, said carbon atom or nitrogenatom forming part of one or more aromatic ring structures wherein atleast one of the ring atoms is nitrogen, oxygen or sulfur, and whereinthe heteroaryl group consists of no atoms other than carbon, hydrogen,aromatic nitrogen, aromatic oxygen and aromatic sulfur. Heteroaryl ringsmay contain 1, 2, 3, or 4 ring atoms selected from are nitrogen, oxygen,and sulfur. If more than one ring is present, the rings may be fused orunfused. As used herein, the term does not preclude the presence of oneor more alkyl, aryl, and/or aralkyl groups (carbon number limitationpermitting) attached to the aromatic ring or aromatic ring system.Non-limiting examples of heteroaryl groups include furanyl, imidazolyl,indolyl, indazolyl (Im), isoxazolyl, methylpyridinyl, oxazolyl,phenylpyridinyl, pyridinyl (pyridyl), pyrrolyl, pyrimidinyl, pyrazinyl,quinolyl, quinazolyl, quinoxalinyl, triazinyl, tetrazolyl, thiazolyl,thienyl, and triazolyl. The term “N-heteroaryl” refers to a heteroarylgroup with a nitrogen atom as the point of attachment. The term“heteroarenediyl” when used without the “substituted” modifier refers toan divalent aromatic group, with two aromatic carbon atoms, two aromaticnitrogen atoms, or one aromatic carbon atom and one aromatic nitrogenatom as the two points of attachment, said atoms forming part of one ormore aromatic ring structure(s) wherein at least one of the ring atomsis nitrogen, oxygen or sulfur, and wherein the divalent group consistsof no atoms other than carbon, hydrogen, aromatic nitrogen, aromaticoxygen and aromatic sulfur. If more than one ring is present, the ringsmay be fused or unfused. Unfused rings may be connected via one or moreof the following: a covalent bond, alkanediyl, or alkenediyl groups(carbon number limitation permitting). As used herein, the term does notpreclude the presence of one or more alkyl, aryl, and/or aralkyl groups(carbon number limitation permitting) attached to the aromatic ring oraromatic ring system. Non-limiting examples of heteroarenediyl groupsinclude:

A “heteroarene” refers to the class of compounds having the formula H—R,wherein R is heteroaryl. Pyridine and quinoline are non-limitingexamples of heteroarenes. When these terms are used with the“substituted” modifier one or more hydrogen atom has been independentlyreplaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH,—OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂,—C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃, —S(O)₂OH, or —S(O)₂NH₂.

The term “heterocycloalkyl” when used without the “substituted” modifierrefers to a monovalent non-aromatic group with a carbon atom or nitrogenatom as the point of attachment, said carbon atom or nitrogen atomforming part of one or more non-aromatic ring structures wherein atleast one of the ring atoms is nitrogen, oxygen or sulfur, and whereinthe heterocycloalkyl group consists of no atoms other than carbon,hydrogen, nitrogen, oxygen and sulfur. Heterocycloalkyl rings maycontain 1, 2, 3, or 4 ring atoms selected from nitrogen, oxygen, orsulfur. If more than one ring is present, the rings may be fused orunfused. As used herein, the term does not preclude the presence of oneor more alkyl groups (carbon number limitation permitting) attached tothe ring or ring system. Also, the term does not preclude the presenceof one or more double bonds in the ring or ring system, provided thatthe resulting group remains non-aromatic. Non-limiting examples ofheterocycloalkyl groups include aziridinyl, azetidinyl, pyrrolidinyl,piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl,tetrahydrofuranyl, tetrahydrothiofuranyl, tetrahydropyranyl, pyranyl,oxiranyl, and oxetanyl. The term “N-heterocycloalkyl” refers to aheterocycloalkyl group with a nitrogen atom as the point of attachment.N-pyrrolidinyl is an example of such a group. The term“heterocycloalkanediyl” when used without the “substituted” modifierrefers to an divalent cyclic group, with two carbon atoms, two nitrogenatoms, or one carbon atom and one nitrogen atom as the two points ofattachment, said atoms forming part of one or more ring structure(s)wherein at least one of the ring atoms is nitrogen, oxygen or sulfur,and wherein the divalent group consists of no atoms other than carbon,hydrogen, nitrogen, oxygen and sulfur. If more than one ring is present,the rings may be fused or unfused. Unfused rings may be connected viaone or more of the following: a covalent bond, alkanediyl, or alkenediylgroups (carbon number limitation permitting). As used herein, the termdoes not preclude the presence of one or more alkyl groups (carbonnumber limitation permitting) attached to the ring or ring system. Also,the term does not preclude the presence of one or more double bonds inthe ring or ring system, provided that the resulting group remainsnon-aromatic. Non-limiting examples of heterocycloalkanediyl groupsinclude:

When these terms are used with the “substituted” modifier one or morehydrogen atom has been independently replaced by —OH, —F, —Cl, —Br, —I,—NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃,—NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃,—NHC(O)CH₃, —S(O)₂OH, or —S(O)₂NH₂.

The term “acyl” when used without the “substituted” modifier refers tothe group —C(O)R, in which R is a hydrogen, alkyl, cycloalkyl, alkenyl,aryl, aralkyl or heteroaryl, as those terms are defined above. Thegroups, —CHO, —C(O)CH₃ (acetyl, Ac), —C(O)CH₂CH₃, —C(O)CH₂CH₂CH₃,—C(O)CH(CH₃)₂, —C(O)CH(CH₂)₂, —C(O)C₆H₅, —C(O)C₆H₄CH₃, —C(O)CH₂C₆H₅,—C(O)(imidazolyl) are non-limiting examples of acyl groups. A “thioacyl”is defined in an analogous manner, except that the oxygen atom of thegroup —C(O)R has been replaced with a sulfur atom, —C(S)R. The term“aldehyde” corresponds to an alkane, as defined above, wherein at leastone of the hydrogen atoms has been replaced with a —CHO group. When anyof these terms are used with the “substituted” modifier one or morehydrogen atom (including a hydrogen atom directly attached to the carbonatom of the carbonyl or thiocarbonyl group, if any) has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂. The groups, —C(O)CH₂CF₃, —CO₂H (carboxyl),—CO₂CH₃ (methylcarboxyl), —CO₂CH₂CH₃, —C(O)NH₂ (carbamoyl), and—CON(CH₃)₂, are non-limiting examples of substituted acyl groups.

The term “alkoxy” when used without the “substituted” modifier refers tothe group —OR, in which R is an alkyl, as that term is defined above.Non-limiting examples include: —OCH₃ (methoxy), —OCH₂CH₃ (ethoxy),—OCH₂CH₂CH₃, —OCH(CH₃)₂(isopropoxy), —OC(CH₃)₃ (tert-butoxy),—OCH(CH₂)₂, —O-cyclopentyl, and —O-cyclohexyl. The terms “cycloalkoxy”,“alkenyloxy”, “alkynyloxy”, “aryloxy”, “aralkoxy”, “heteroaryloxy”,“heterocycloalkoxy”, and “acyloxy”, when used without the “substituted”modifier, refers to groups, defined as —OR, in which R is cycloalkyl,alkenyl, alkynyl, aryl, aralkyl, heteroaryl, heterocycloalkyl, and acyl,respectively. The term “alkoxydiyl” refers to the divalent group—O-alkanediyl-, —O-alkanediyl-O—, or -alkanediyl-O-alkanediyl-. The term“alkylthio” and “acylthio” when used without the “substituted” modifierrefers to the group —SR, in which R is an alkyl and acyl, respectively.The term “alcohol” corresponds to an alkane, as defined above, whereinat least one of the hydrogen atoms has been replaced with a hydroxygroup. The term “ether” corresponds to an alkane, as defined above,wherein at least one of the hydrogen atoms has been replaced with analkoxy group. When any of these terms is used with the “substituted”modifier one or more hydrogen atom has been independently replaced by—OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H, —CO₂CH₃, —CN, —SH, —OCH₃,—OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃, —N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃,—C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃, —S(O)₂OH, or —S(O)₂NH₂.

The term “alkylamino” when used without the “substituted” modifierrefers to the group —NHR, in which R is an alkyl, as that term isdefined above. Non-limiting examples include: —NHCH₃ and —NHCH₂CH₃. Theterm “dialkylamino” when used without the “substituted” modifier refersto the group —NRR′, in which R and R′ can be the same or different alkylgroups, or R and R′ can be taken together to represent an alkanediyl.Non-limiting examples of dialkylamino groups include: —N(CH₃)₂ and—N(CH₃)(CH₂CH₃). The terms “cycloalkylamino”, “alkenylamino”,“alkynylamino”, “arylamino”, “aralkylamino”, “heteroarylamino”,“heterocycloalkylamino”, “alkoxyamino”, and “alkylsulfonylamino” whenused without the “substituted” modifier, refers to groups, defined as—NHR, in which R is cycloalkyl, alkenyl, alkynyl, aryl, aralkyl,heteroaryl, heterocycloalkyl, alkoxy, and alkylsulfonyl, respectively. Anon-limiting example of an arylamino group is —NHC₆H₅. The term“alkylaminodiyl” refers to the divalent group —NH-alkanediyl-,—NH-alkanediyl-NH—, or -alkanediyl-NH-alkanediyl-. The term “amido”(acylamino), when used without the “substituted” modifier, refers to thegroup —NHR, in which R is acyl, as that term is defined above. Anon-limiting example of an amido group is —NHC(O)CH₃. The term“alkylimino” when used without the “substituted” modifier refers to thedivalent group ═NR, in which R is an alkyl, as that term is definedabove. When any of these terms is used with the “substituted” modifierone or more hydrogen atom attached to a carbon atom has beenindependently replaced by —OH, —F, —Cl, —Br, —I, —NH₂, —NO₂, —CO₂H,—CO₂CH₃, —CN, —SH, —OCH₃, —OCH₂CH₃, —C(O)CH₃, —NHCH₃, —NHCH₂CH₃,—N(CH₃)₂, —C(O)NH₂, —C(O)NHCH₃, —C(O)N(CH₃)₂, —OC(O)CH₃, —NHC(O)CH₃,—S(O)₂OH, or —S(O)₂NH₂. The groups —NHC(O)OCH₃ and —NHC(O)NHCH₃ arenon-limiting examples of substituted amido groups.

The use of the word “a” or “an,” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

As used in this application, the term “average molecular weight” refersto the relationship between the number of moles of each polymer speciesand the molar mass of that species. In particular, each polymer moleculemay have different levels of polymerization and thus a different molarmass. The average molecular weight can be used to represent themolecular weight of a plurality of polymer molecules. Average molecularweight is typically synonymous with average molar mass. In particular,there are three major types of average molecular weight: number averagemolar mass, weight (mass) average molar mass, and Z-average molar mass.In the context of this application, unless otherwise specified, theaverage molecular weight represents either the number average molar massor weight average molar mass of the formula. In some embodiments, theaverage molecular weight is the number average molar mass. In someembodiments, the average molecular weight may be used to describe a PEGcomponent present in a lipid.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and also covers other unlisted steps.

The term “effective,” as that term is used in the specification and/orclaims, means adequate to accomplish a desired, expected, or intendedresult. “Effective amount,” “Therapeutically effective amount” or“pharmaceutically effective amount” when used in the context of treatinga patient or subject with a compound means that amount of the compoundwhich, when administered to a subject or patient for treating a disease,is sufficient to effect such treatment for the disease.

As used herein, the term “IC₅₀” refers to an inhibitory dose which is50% of the maximum response obtained. This quantitative measureindicates how much of a particular drug or other substance (inhibitor)is needed to inhibit a given biological, biochemical or chemical process(or component of a process, i.e. an enzyme, cell, cell receptor ormicroorganism) by half.

An “isomer” of a first compound is a separate compound in which eachmolecule contains the same constituent atoms as the first compound, butwhere the configuration of those atoms in three dimensions differs.

As used herein, the term “patient” or “subject” refers to a livingmammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat,mouse, rat, guinea pig, or transgenic species thereof. In certainembodiments, the patient or subject is a primate. Non-limiting examplesof human subjects are adults, juveniles, infants and fetuses.

As generally used herein “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues, organs, and/or bodily fluids of human beings andanimals without excessive toxicity, irritation, allergic response, orother problems or complications commensurate with a reasonablebenefit/risk ratio.

“Pharmaceutically acceptable salts” means salts of compounds of thepresent invention which are pharmaceutically acceptable, as definedabove, and which possess the desired pharmacological activity. Suchsalts include acid addition salts formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or with organic acids such as1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid,2-naphthalenesulfonic acid, 3-phenylpropionic acid,4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylic acid),4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid,aliphatic mono- and dicarboxylic acids, aliphatic sulfuric acids,aromatic sulfuric acids, benzenesulfonic acid, benzoic acid,camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid,cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid,glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid,heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid,laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelicacid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoicacid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substitutedalkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid,salicylic acid, stearic acid, succinic acid, tartaric acid,tertiarybutylacetic acid, trimethylacetic acid, and the like.Pharmaceutically acceptable salts also include base addition salts whichmay be formed when acidic protons present are capable of reacting withinorganic or organic bases. Acceptable inorganic bases include sodiumhydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide andcalcium hydroxide. Acceptable organic bases include ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine and thelike. It should be recognized that the particular anion or cationforming a part of any salt of this invention is not critical, so long asthe salt, as a whole, is pharmacologically acceptable. Additionalexamples of pharmaceutically acceptable salts and their methods ofpreparation and use are presented in Handbook of Pharmaceutical Salts:Properties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag HelveticaChimica Acta, 2002).

The term “pharmaceutically acceptable carrier,” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, solvent or encapsulatingmaterial, involved in carrying or transporting a chemical agent.

“Prevention” or “preventing” includes: (1) inhibiting the onset of adisease in a subject or patient which may be at risk and/or predisposedto the disease but does not yet experience or display any or all of thepathology or symptomatology of the disease, and/or (2) slowing the onsetof the pathology or symptomatology of a disease in a subject or patientwhich may be at risk and/or predisposed to the disease but does not yetexperience or display any or all of the pathology or symptomatology ofthe disease.

A “repeat unit” is the simplest structural entity of certain materials,for example, frameworks and/or polymers, whether organic, inorganic ormetal-organic. In the case of a polymer chain, repeat units are linkedtogether successively along the chain, like the beads of a necklace. Forexample, in polyethylene, —[—CH₂CH₂—]_(n)—, the repeat unit is —CH₂CH₂—.The subscript “n” denotes the degree of polymerization, that is, thenumber of repeat units linked together. When the value for “n” is leftundefined or where “n” is absent, it simply designates repetition of theformula within the brackets as well as the polymeric nature of thematerial. The concept of a repeat unit applies equally to where theconnectivity between the repeat units extends three dimensionally, suchas in metal organic frameworks, modified polymers, thermosettingpolymers, etc. Within the context of the dendrimer, the repeating unitmay also be described as the branching unit, interior layers, orgenerations. Similarly, the terminating group may also be described asthe surface group.

A “stereoisomer” or “optical isomer” is an isomer of a given compound inwhich the same atoms are bonded to the same other atoms, but where theconfiguration of those atoms in three dimensions differs. “Enantiomers”are stereoisomers of a given compound that are mirror images of eachother, like left and right hands. “Diastereomers” are stereoisomers of agiven compound that are not enantiomers. Chiral molecules contain achiral center, also referred to as a stereocenter or stereogenic center,which is any point, though not necessarily an atom, in a moleculebearing groups such that an interchanging of any two groups leads to astereoisomer. In organic compounds, the chiral center is typically acarbon, phosphorus or sulfur atom, though it is also possible for otheratoms to be stereocenters in organic and inorganic compounds. A moleculecan have multiple stereocenters, giving it many stereoisomers. Incompounds whose stereoisomerism is due to tetrahedral stereogeniccenters (e.g., tetrahedral carbon), the total number of hypotheticallypossible stereoisomers will not exceed 2^(n), where n is the number oftetrahedral stereocenters. Molecules with symmetry frequently have fewerthan the maximum possible number of stereoisomers. A 50:50 mixture ofenantiomers is referred to as a racemic mixture. Alternatively, amixture of enantiomers can be enantiomerically enriched so that oneenantiomer is present in an amount greater than 50%. Typically,enantiomers and/or diastereomers can be resolved or separated usingtechniques known in the art. It is contemplated that that for anystereocenter or axis of chirality for which stereochemistry has not beendefined, that stereocenter or axis of chirality can be present in its Rform, S form, or as a mixture of the R and S forms, including racemicand non-racemic mixtures. As used herein, the phrase “substantially freefrom other stereoisomers” means that the composition contains ≤15%, morepreferably ≤10%, even more preferably ≤5%, or most preferably ≤1% ofanother stereoisomer(s).

“Treatment” or “treating” includes (1) inhibiting a disease in a subjector patient experiencing or displaying the pathology or symptomatology ofthe disease (e.g., arresting further development of the pathology and/orsymptomatology), (2) ameliorating a disease in a subject or patient thatis experiencing or displaying the pathology or symptomatology of thedisease (e.g., reversing the pathology and/or symptomatology), and/or(3) effecting any measurable decrease in a disease in a subject orpatient that is experiencing or displaying the pathology orsymptomatology of the disease.

The above definitions supersede any conflicting definition in anyreference that is incorporated by reference herein. The fact thatcertain terms are defined, however, should not be considered asindicative that any term that is undefined is indefinite. Rather, allterms used are believed to describe the invention in terms such that oneof ordinary skill can appreciate the scope and practice the presentinvention.

B. DENDRIMERS AND DENDRITIC STRUCTURES

In some aspects of the present disclosure, dendrimers containinglipophilic and cationic components are provided. Dendrimers are apolymer exhibiting regular dendritic branching, formed by the sequentialor generational addition of branched layers to or from a core and arecharacterized by a core, at least one interior branched layer, and asurface branched layer. (See Petar R. Dvornic and Donald A. Tomalia inChem. in Britain, 641-645, August 1994.) In other embodiments, the term“dendrimer” as used herein is intended to include, but is not limitedto, a molecular architecture with an interior core, interior layers (or“generations”) of repeating units regularly attached to this initiatorcore, and an exterior surface of terminal groups attached to theoutermost generation. A “dendron” is a species of dendrimer havingbranches emanating from a focal point which is or can be joined to acore, either directly or through a linking moiety to form a largerdendrimer. In some embodiments, the dendrimer structures have radiatingrepeating groups from a central core which doubles with each repeatingunit for each branch. In some embodiments, the dendrimers describedherein may be described as a small molecule, medium-sized molecules,lipids, or lipid-like material. These terms may be used to describedcompounds described herein which have a dendron like appearance (e.g.molecules which radiate from a single focal point).

While dendrimers are polymers, dendrimers are preferable to traditionalpolymers because they have a controllable structure, a single molecularweight, numerous and controllable surface functionalities, andtraditionally adopt a globular conformation after reaching a specificgeneration. Dendrimers can be prepared by sequentially reactions of eachrepeating unit to produce monodisperse, tree-like and/or generationalstructure polymeric structures. Individual dendrimers consist of acentral core molecule, with a dendritic wedge attached to one or morefunctional sites on that central core. The dendrimeric surface layer canhave a variety of functional groups disposed thereon including anionic,cationic, hydrophilic, or lipophilic groups, according to the assemblymonomers used during the preparation.

Modifying the functional groups and/or the chemical properties of thecore, repeating units, and the surface or terminating groups, theirphysical properties can be modulated. Some properties which can bevaried include, but are not limited to, solubility, toxicity,immunogenicity and bioattachment capability. Dendrimers are oftendescribed by their generation or number of repeating units in thebranches. A dendrimer consisting of only the core molecule is referredto as Generation 0, while each consecutive repeating unit along allbranches is Generation 1, Generation 2, and so on until the terminatingor surface group. In some embodiments, half generations are possibleresulting from only the first condensation reaction with the amine andnot the second condensation reaction with the thiol

Preparation of dendrimers requires a level of synthetic control achievedthrough series of stepwise reactions comprising building the dendrimerby each consecutive group. Dendrimer synthesis can be of the convergentor divergent type. During divergent dendrimer synthesis, the molecule isassembled from the core to the periphery in a stepwise process involvingattaching one generation to the previous and then changing functionalgroups for the next stage of reaction.

Functional group transformation is necessary to prevent uncontrolledpolymerization. Such polymerization would lead to a highly branchedmolecule that is not monodisperse and is otherwise known as ahyperbranched polymer. Due to steric effects, continuing to reactdendrimer repeat units leads to a sphere shaped or globular molecule,until steric overcrowding prevents complete reaction at a specificgeneration and destroys the molecule's monodispersity. Thus, in someembodiments, the dendrimers of G1-G10 generation are specificallycontemplated. In some embodiments, the dendrimers comprise 1, 2, 3, 4,5, 6, 7, 8, 9, or 10 repeating units, or any range derivable therein. Insome embodiments, the dendrimers used herein are G0, G1, G2, or G3.However, the number of possible generations (such as 11, 12, 13, 14, 15,20, or 25) may be increased by reducing the spacing units in thebranching polymer.

Additionally, dendrimers have two major chemical environments: theenvironment created by the specific surface groups on the terminationgeneration and the interior of the dendritic structure which due to thehigher order structure can be shielded from the bulk media and thesurface groups. Because of these different chemical environments,dendrimers have found numerous different potential uses including intherapeutic applications.

In some aspects, the dendrimers of the present disclosure are assembledusing the differential reactivity of the acrylate and methacrylategroups with amines and thiols. The dendrimers that may be used hereininclude secondary or tertiary amines and thioethers formed by thereaction of an acrylate group with a primary or secondary amine and amethacrylate with a mercapto group. Additionally, the repeating units ofthe dendrimers described herein may contain groups which are degradableunder physiological conditions. In some embodiments, these repeatingunits may contain one or more germinal diethers, esters, amides, ordisulfides groups. In some embodiments, the core molecule is a monoaminewhich allows dendritic polymerization in only one direction. In otherembodiments, the core molecule is a polyamine with multiple differentdendritic branches which each may comprise one or more repeating units.The dendrimer may be formed by removing one or more hydrogen atoms fromthis core. In some embodiments, these hydrogen atoms are on a heteroatomsuch as a nitrogen atom. In some embodiments, the terminating group is alipophilic groups such as a long chain alkyl or alkenyl group. In otherembodiments, the terminating group is a long chain haloalkyl orhaloalkenyl group. In other embodiments, the terminating group is analiphatic or aromatic group containing an ionizable group such as anamine (—NH₂) or a carboxylic acid (—CO₂H). In still other embodiments,the terminating group is an aliphatic or aromatic group containing oneor more hydrogen bond donors such as a hydroxide group, an amide group,or an ester.

The dendrimers provided by the present disclosure are shown, forexample, above in the summary of the invention section and in the claimsbelow. They may be made using the methods outlined in the Examplessection. These methods can be further modified and optimized using theprinciples and techniques of organic chemistry as applied by a personskilled in the art. Such principles and techniques are taught, forexample, in March's Advanced Organic Chemistry: Reactions, Mechanisms,and Structure (2007), which is incorporated by reference herein.

The dendrimers of the present disclosure may contain one or moreasymmetrically-substituted carbon or nitrogen atoms, and may be isolatedin optically active or racemic form. Thus, all chiral, diastereomeric,racemic form, epimeric form, and all geometric isomeric forms of achemical formula are intended, unless the specific stereochemistry orisomeric form is specifically indicated. Dendrimers may occur asracemates and racemic mixtures, single enantiomers, diastereomericmixtures and individual diastereomers. In some embodiments, a singlediastereomer is obtained. The chiral centers of the dendrimers of thepresent disclosure can have the S or the R configuration. Furthermore,it is contemplated that one or more of the dendrimers may be present asconstitutional isomers. In some embodiments, the compounds have the sameformula but different connectivity to the nitrogen atoms of the core.Without wishing to be bound by any theory, it is believed that suchdendrimers exist because the starting monomers react first with theprimary amines and then statistically with any secondary amines present.Thus, the constitutional isomers may present the fully reacted primaryamines and then a mixture of reacted secondary amines.

Chemical formulas used to represent dendrimers of the present disclosurewill typically only show one of possibly several different tautomers.For example, many types of ketone groups are known to exist inequilibrium with corresponding enol groups. Similarly, many types ofimine groups exist in equilibrium with enamine groups. Regardless ofwhich tautomer is depicted for a given formula, and regardless of whichone is most prevalent, all tautomers of a given chemical formula areintended.

The dendrimers of the present disclosure may also have the advantagethat they may be more efficacious than, be less toxic than, be longeracting than, be more potent than, produce fewer side effects than, bemore easily absorbed than, and/or have a better pharmacokinetic profile(e.g., higher oral bioavailability and/or lower clearance) than, and/orhave other useful pharmacological, physical, or chemical propertiesover, compounds known in the prior art, whether for use in theindications stated herein or otherwise.

In addition, atoms making up the dendrimers of the present disclosureare intended to include all isotopic forms of such atoms. Isotopes, asused herein, include those atoms having the same atomic number butdifferent mass numbers. By way of general example and withoutlimitation, isotopes of hydrogen include tritium and deuterium, andisotopes of carbon include ¹³C and ¹⁴C.

It should be recognized that the particular anion or cation forming apart of any salt form of a dendrimer provided herein is not critical, solong as the salt, as a whole, is pharmacologically acceptable.Additional examples of pharmaceutically acceptable salts and theirmethods of preparation and use are presented in Handbook ofPharmaceutical Salts: Properties, and Use (2002), which is incorporatedherein by reference.

C. HELPER LIPIDS

In some aspects of the present disclosure, one or more helper lipids aremixed with the polymers of the instant disclosure to create acomposition. In some embodiments, the polymers are mixed with 1, 2, 3,4, or 5 different types of helper lipids. It is contemplated that thepolymers can be mixed with multiple different lipids of a single type.In some embodiments, the lipid could be a steroid or a steroidderivative. In other embodiments, the lipid is a PEG lipid. In otherembodiments, the lipid is a phospholipid. In other embodiments, thedendrimer composition comprises a steroid or a steroid derivative, a PEGlipid, and a phospholipid.

1. Steroids and Steroid Derivatives

In some aspects of the present disclosure, the polymers are mixed withone or more steroid or a steroid derivative to create a dendrimercomposition. In some embodiments, the steroid or steroid derivativecomprises any steroid or steroid derivative. As used herein, in someembodiments, the term “steroid” is a class of compounds with a four ring17 carbon cyclic structure which can further comprises one or moresubstitutions including alkyl groups, alkoxy groups, hydroxy groups, oxogroups, acyl groups, or a double bond between two or more carbon atoms.In one aspect, the ring structure of a steroid comprises three fusedcyclohexyl rings and a fused cyclopentyl ring as shown in the formulabelow:

In some embodiments, a steroid derivative comprises the ring structureabove with one or more non-alkyl substitutions. In some embodiments, thesteroid or steroid derivative is a sterol wherein the formula is furtherdefined as:

In some embodiments of the present disclosure, the steroid or steroidderivative is a cholestane or cholestane derivative. In a cholestane,the ring structure is further defined by the formula:

As described above, a cholestane derivative includes one or morenon-alkyl substitution of the above ring system. In some embodiments,the cholestane or cholestane derivative is a cholestene or cholestenederivative or a sterol or a sterol derivative. In other embodiments, thecholestane or cholestane derivative is both a cholestere and a sterol ora derivative thereof.

In some embodiments, the compositions may further comprise a molar ratioof the steroid to the dendrimer from about 1:10 to about 1:20. In someembodiments, the molar ratio is from about 1:20, 1:18, 1:16, 1:14, 1:12,1:10, 1:8, 1:6, 1:4, 1:2, 1:1, 2:1, 4:1, 6:1, 8:1, to about 10:1 or anyrange derivable therein. In some embodiments, the molar ratio is about38:50 or about 1:5.

2. PEG or PEGylated Lipid

In some aspects of the present disclosure, the polymers are mixed withone or more PEGylated lipids (or PEG lipid) to create a dendrimercomposition. In some embodiments, the present disclosure comprises usingany lipid to which a PEG group has been attached. In some embodiments,the PEG lipid is a diglyceride which also comprises a PEG chain attachedto the glycerol group. In other embodiments, the PEG lipid is a compoundwhich contains one or more C6-C24 long chain alkyl or alkenyl group or aC6-C24 fatty acid group attached to a linker group with a PEG chain.Some non-limiting examples of a PEG lipid includes a PEG modifiedphosphatidylethanolamine and phosphatidic acid, a PEG ceramideconjugated, PEG modified dialkylamines and PEG modified1,2-diacyloxypropan-3-amines, PEG modified diacylglycerols anddialkylglycerols. In some embodiments, PEG modifieddiastearoylphosphatidylethanolamine or PEG modifieddimyristoyl-sn-glycerol. In some embodiments, the PEG modification ismeasured by the molecular weight of PEG component of the lipid. In someembodiments, the PEG modification has a molecular weight from about 100to about 15,000. In some embodiments, the molecular weight is from about200 to about 500, from about 400 to about 5,000, from about 500 to about3,000, or from about 1,200 to about 3,000. The molecular weight of thePEG modification is from about 100, 200, 400, 500, 600, 800, 1,000,1,250, 1,500, 1,750, 2,000, 2,250, 2,500, 2,750, 3,000, 3,500, 4,000,4,500, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 12,500, to about15,000. Some non-limiting examples of lipids that may be used in thepresent invention are taught by U.S. Pat. No. 5,820,873, WO 2010/141069,or U.S. Pat. No. 8,450,298, which is incorporated herein by reference.

In another aspect, the PEG lipid has the formula:

wherein: R₁₂ and R₁₃ are each independently alkyl_((C≤24)),alkenyl_((C≤24)), or a substituted version of either of these groups;R_(e) is hydrogen, alkyl_((C≤8)), or substituted alkyl_((C≤8)); and x is1-250. In some embodiments, R_(e) is alkyl_((C≤8)) such as methyl. R₁₂and R₁₃ are each independently alkyl_((C≤4-20)). In some embodiments, xis 5-250. In one embodiment, x is 5-125 or x is 100-250. In someembodiments, the PEG lipid is 1,2-dimyristoyl-sn-glycerol,methoxypolyethylene glycol.

In another aspect, the PEG lipid has the formula:

wherein: n₁ is an integer between 1 and 100 and n₂ and n₃ are eachindependently selected from an integer between 1 and 29. In someembodiments, n₁ is 5, 10, 15, 20, 25, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, or 100, or any range derivable therein. In someembodiments, n₁ is from about 30 to about 50. In some embodiments, n₂ isfrom 5 to 23. In some embodiments, n₂ is 11 to about 17. In someembodiments, n₃ is from 5 to 23. In some embodiments, n₃ is 11 to about17.

In some embodiments, the compositions may further comprise a molar ratioof the PEG lipid to the dendrimer from about 1:1 to about 1:250. In someembodiments, the molar ratio is from about 1:1, 1:10, 1:20, 1:30, 1:40,1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:110, 1:120, 1:125, 1:150, 1:175,1:200, 1:225, to about 1:250 or any range derivable therein. In someembodiments, the molar ratio is about 1:25 or about 3:100.

3. Phospholipid

In some aspects of the present disclosure, the polymers are mixed withone or more phospholipids to create a dendrimer composition. In someembodiments, any lipid which also comprises a phosphate group. In someembodiments, the phospholipid is a structure which contains one or twolong chain C6-C24 alkyl or alkenyl groups, a glycerol or a sphingosine,one or two phosphate groups, and, optionally, a small organic molecule.In some embodiments, the small organic molecule is an amino acid, asugar, or an amino substituted alkoxy group, such as choline orethanolamine. In some embodiments, the phospholipid is aphosphatidylcholine. In some embodiments, the phospholipid isdistearoylphosphatidylcholine or dioleoylphosphatidylethanolamine.

In some embodiments, the compositions may further comprise a molar ratioof the phospholipid to the dendrimer from about 1:10 to about 1:20. Insome embodiments, the molar ratio is from about 1:20, 1:18, 1:16, 1:14,1:12, 1:10, 1:8, 1:6, 1:4, 1:2, 1:1, 2:1, 4:1, 6:1, 8:1, to about 10:1or any range derivable therein. In some embodiments, the molar ratio isabout 38:50 or about 1:5.

D. NUCLEIC ACIDS AND NUCLEIC ACID BASED THERAPEUTIC AGENTS

1. Nucleic Acids

In some aspects of the present disclosure, the dendrimer compositionscomprise one or more nucleic acids. In some embodiments, the dendrimercomposition comprises one or more nucleic acids present in a weightratio to the dendrimer from about 5:1 to about 1:100. In someembodiments, the weight ratio of nucleic acid to dendrimer is from about5:1, 2.5:1, 1:1, 1:5, 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45,1:50, 1:60, 1:70, 1:80, 1:90, or 1:100, or any range derivable therein.In some embodiments, the weight ratio is about 1:25 or about 1:7. Inaddition, it should be clear that the present disclosure is not limitedto the specific nucleic acids disclosed herein. The present invention isnot limited in scope to any particular source, sequence, or type ofnucleic acid, however, as one of ordinary skill in the art could readilyidentify related homologs in various other sources of the nucleic acidincluding nucleic acids from non-human species (e.g., mouse, rat,rabbit, dog, monkey, gibbon, chimp, ape, baboon, cow, pig, horse, sheep,cat and other species). It is contemplated that the nucleic acid used inthe present disclosure can comprises a sequence based upon anaturally-occurring sequence. Allowing for the degeneracy of the geneticcode, sequences that have at least about 50%, usually at least about60%, more usually about 70%, most usually about 80%, preferably at leastabout 90% and most preferably about 95% of nucleotides that areidentical to the nucleotide sequence of the naturally-occurringsequence. In another embodiment, the nucleic acid is a complementarysequence to a naturally occurring sequence, or complementary to 75%,80%, 85%, 90%, 95% and 100%.

In some aspects, the nucleic acid is a sequence which silences, iscomplimentary to, or replaces another sequence present in vivo.Sequences of 17 bases in length should occur only once in the humangenome and, therefore, suffice to specify a unique target sequence.Although shorter oligomers are easier to make and increase in vivoaccessibility, numerous other factors are involved in determining thespecificity of hybridization. Both binding affinity and sequencespecificity of an oligonucleotide to its complementary target increaseswith increasing length. It is contemplated that exemplaryoligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or morebase pairs will be used, although others are contemplated. Longerpolynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000 orlonger are contemplated as well.

The nucleic acid used herein may be derived from genomic DNA, i.e.,cloned directly from the genome of a particular organism. In preferredembodiments, however, the nucleic acid would comprise complementary DNA(cDNA). Also contemplated is a cDNA plus a natural intron or an intronderived from another gene; such engineered molecules are sometimereferred to as “mini-genes.” At a minimum, these and other nucleic acidsof the present invention may be used as molecular weight standards in,for example, gel electrophoresis.

The term “cDNA” is intended to refer to DNA prepared using messenger RNA(mRNA) as template. The advantage of using a cDNA, as opposed to genomicDNA or DNA polymerized from a genomic, non- or partially-processed RNAtemplate, is that the cDNA primarily contains coding sequences of thecorresponding protein. There may be times when the full or partialgenomic sequence is preferred, such as where the non-coding regions arerequired for optimal expression or where non-coding regions such asintrons are to be targeted in an antisense strategy.

In some embodiments, the nucleic acid comprises one or more antisensesegments which inhibits expression of a gene or gene product. Antisensemethodology takes advantage of the fact that nucleic acids tend to pairwith “complementary” sequences. By complementary, it is meant thatpolynucleotides are those which are capable of base-pairing according tothe standard Watson-Crick complementarity rules. That is, the largerpurines will base pair with the smaller pyrimidines to form combinationsof guanine paired with cytosine (G:C) and adenine paired with eitherthymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) inthe case of RNA. Inclusion of less common bases such as inosine,5-methylcytosine, 6-methyladenine, hypoxanthine and others inhybridizing sequences does not interfere with pairing.

Targeting double-stranded (ds) DNA with polynucleotides leads totriple-helix formation; targeting RNA will lead to double-helixformation. Antisense polynucleotides, when introduced into a targetcell, specifically bind to their target polynucleotide and interferewith transcription, RNA processing, transport, translation and/orstability. Antisense RNA constructs, or DNA encoding such antisenseRNA's, may be employed to inhibit gene transcription or translation orboth within a host cell, either in vitro or in vivo, such as within ahost animal, including a human subject.

Antisense constructs may be designed to bind to the promoter and othercontrol regions, exons, introns or even exon-intron boundaries of agene. It is contemplated that the most effective antisense constructswill include regions complementary to intron/exon splice junctions.Thus, it is proposed that a preferred embodiment includes an antisenseconstruct with complementarity to regions within 50-200 bases of anintron-exon splice junction. It has been observed that some exonsequences can be included in the construct without seriously affectingthe target selectivity thereof. The amount of exonic material includedwill vary depending on the particular exon and intron sequences used.One can readily test whether too much exon DNA is included simply bytesting the constructs in vitro to determine whether normal cellularfunction is affected or whether the expression of related genes havingcomplementary sequences is affected.

As stated above, “complementary” or “antisense” means polynucleotidesequences that are substantially complementary over their entire lengthand have very few base mismatches. For example, sequences of fifteenbases in length may be termed complementary when they have complementarynucleotides at thirteen or fourteen positions. Naturally, sequenceswhich are completely complementary will be sequences which are entirelycomplementary throughout their entire length and have no basemismatches. Other sequences with lower degrees of homology also arecontemplated. For example, an antisense construct which has limitedregions of high homology, but also contains a non-homologous region(e.g., ribozyme; see below) could be designed. These molecules, thoughhaving less than 50% homology, would bind to target sequences underappropriate conditions.

It may be advantageous to combine portions of genomic DNA with cDNA orsynthetic sequences to form a siRNA or to generate specific constructs.For example, where an intron is desired in the ultimate construct, agenomic clone will need to be used. The cDNA, siRNA, or a synthesizedpolynucleotide may provide more convenient restriction sites for theremaining portion of the construct and, therefore, would be used for therest of the sequence. Other embodiments include dsRNA or ssRNA, whichmay be used to target genomic sequences or coding/non-codingtranscripts.

In other embodiments, the dendrimer compositions may comprise a nucleicacid which comprises one or more expression vectors are used in a genetherapy. Expression requires that appropriate signals be provided in thevectors, and which include various regulatory elements, such asenhancers/promoters from both viral and mammalian sources that driveexpression of the genes of interest in host cells. Elements designed tooptimize messenger RNA stability and translatability in host cells alsoare defined. The conditions for the use of a number of dominant drugselection markers for establishing permanent, stable cell clonesexpressing the products are also provided, as is an element that linksexpression of the drug selection markers to expression of thepolypeptide.

Throughout this application, the term “expression construct” is meant toinclude any type of genetic construct containing a nucleic acid codingfor a gene product in which part or all of the nucleic acid encodingsequence is capable of being transcribed. The transcript may betranslated into a protein, but it need not be. In certain embodiments,expression includes both transcription of a gene and translation of mRNAinto a gene product. In other embodiments, expression only includestranscription of the nucleic acid encoding a gene of interest.

The term “vector” is used to refer to a carrier nucleic acid moleculeinto which a nucleic acid sequence can be inserted for introduction intoa cell where it can be replicated. A nucleic acid sequence can be“exogenous,” which means that it is foreign to the cell into which thevector is being introduced or that the sequence is homologous to asequence in the cell but in a position within the host cell nucleic acidin which the sequence is ordinarily not found. Vectors include plasmids,cosmids, viruses (bacteriophage, animal viruses, and plant viruses), andartificial chromosomes (e.g., YACs). One of skill in the art would bewell equipped to construct a vector through standard recombinanttechniques, which are described in Sambrook et al. (1989) and Ausubel etal. (1994), both incorporated herein by reference.

The term “expression vector” refers to a vector containing a nucleicacid sequence coding for at least part of a gene product capable ofbeing transcribed. In some cases, RNA molecules are then translated intoa protein, polypeptide, or peptide. In other cases, these sequences arenot translated, for example, in the production of antisense molecules orribozymes. Expression vectors can contain a variety of “controlsequences,” which refer to nucleic acid sequences necessary for thetranscription and possibly translation of an operably linked codingsequence in a particular host organism. In addition to control sequencesthat govern transcription and translation, vectors and expressionvectors may contain nucleic acid sequences that serve other functions aswell and are described infra.

2. siRNA

As mentioned above, the present invention contemplates the use of one ormore inhibitory nucleic acid for reducing expression and/or activationof a gene or gene product. Examples of an inhibitory nucleic acidinclude but are not limited to molecules targeted to an nucleic acidsequence, such as an siRNA (small interfering RNA), short hairpin RNA(shRNA), double-stranded RNA, an antisense oligonucleotide, a ribozymeand molecules targeted to a gene or gene product such as an aptamer.

An inhibitory nucleic acid may inhibit the transcription of a gene orprevent the translation of the gene transcript in a cell. An inhibitorynucleic acid may be from 16 to 1000 nucleotides long, and in certainembodiments from 18 to 100 nucleotides long.

Inhibitory nucleic acids are well known in the art. For example, siRNA,shRNA and double-stranded RNA have been described in U.S. Pat. Nos.6,506,559 and 6,573,099, as well as in U.S. Patent Publications2003/0051263, 2003/0055020, 2004/0265839, 2002/0168707, 2003/0159161,and 2004/0064842, all of which are herein incorporated by reference intheir entirety.

Since the discovery of RNAi by Fire and colleagues in 1998, thebiochemical mechanisms have been rapidly characterized. Double strandedRNA (dsRNA) is cleaved by Dicer, which is an RNAase III familyribonuclease. This process yields siRNAs of ˜21 nucleotides in length.These siRNAs are incorporated into a multiprotein RNA-induced silencingcomplex (RISC) that is guided to target mRNA. RISC cleaves the targetmRNA in the middle of the complementary region. In mammalian cells, therelated microRNAs (miRNAs) are found that are short RNA fragments (˜22nucleotides). miRNAs are generated after Dicer-mediated cleavage oflonger (˜70 nucleotide) precursors with imperfect hairpin RNAstructures. The miRNA is incorporated into a miRNA-protein complex(miRNP), which leads to translational repression of target mRNA.

In designing a nucleic acid capable of generating an RNAi effect, thereare several factors that need to be considered such as the nature of thesiRNA, the durability of the silencing effect, and the choice ofdelivery system. To produce an RNAi effect, the siRNA that is introducedinto the organism will typically contain exonic sequences. Furthermore,the RNAi process is homology dependent, so the sequences must becarefully selected so as to maximize gene specificity, while minimizingthe possibility of cross-interference between homologous, but notgene-specific sequences. Particularly the siRNA exhibits greater than80, 85, 90, 95, 98% or even 100% identity between the sequence of thesiRNA and a portion of a EphA nucleotide sequence. Sequences less thanabout 80% identical to the target gene are substantially less effective.Thus, the greater identity between the siRNA and the gene to beinhibited, the less likely expression of unrelated genes will beaffected.

In addition, the size of the siRNA is an important consideration. Insome embodiments, the present disclosure relates to siRNA molecules thatinclude at least about 19-25 nucleotides, and are able to modulate geneexpression. In the context of the present disclosure, the siRNA isparticularly less than 500, 200, 100, 50, 25, or 20 nucleotides inlength. In some embodiments, the siRNA is from about 25 nucleotides toabout 35 nucleotides or from about 19 nucleotides to about 25nucleotides in length.

To improve the effectiveness of siRNA-mediated gene silencing,guidelines for selection of target sites on mRNA have been developed foroptimal design of siRNA (Soutschek et al., 2004; Wadhwa et al., 2004).These strategies may allow for rational approaches for selecting siRNAsequences to achieve maximal gene knockdown. To facilitate the entry ofsiRNA into cells and tissues, a variety of vectors including plasmidsand viral vectors such as adenovirus, lentivirus, and retrovirus havebeen used (Wadhwa et al., 2004).

Within an inhibitory nucleic acid, the components of a nucleic acid neednot be of the same type or homogenous throughout (e.g., an inhibitorynucleic acid may comprise a nucleotide and a nucleic acid or nucleotideanalog). Typically, an inhibitory nucleic acid form a double-strandedstructure; the double-stranded structure may result from two separatenucleic acids that are partially or completely complementary. In certainembodiments of the present invention, the inhibitory nucleic acid maycomprise only a single nucleic acid (polynucleotide) or nucleic acidanalog and form a double-stranded structure by complementing with itself(e.g., forming a hairpin loop). The double-stranded structure of theinhibitory nucleic acid may comprise 16-500 or more contiguousnucleobases, including all ranges derivable thereof. The inhibitorynucleic acid may comprise 17 to 35 contiguous nucleobases, moreparticularly 18 to 30 contiguous nucleobases, more particularly 19 to 25nucleobases, more particularly 20 to 23 contiguous nucleobases, or 20 to22 contiguous nucleobases, or 21 contiguous nucleobases that hybridizewith a complementary nucleic acid (which may be another part of the samenucleic acid or a separate complementary nucleic acid) to form adouble-stranded structure.

siRNA can be obtained from commercial sources, natural sources, or canbe synthesized using any of a number of techniques well-known to thoseof ordinary skill in the art. For example, commercial sources ofpredesigned siRNA include Invitrogen's Stealth™ Select technology(Carlsbad, Calif.), Ambion® (Austin, Tex.), and Qiagen® (Valencia,Calif.). An inhibitory nucleic acid that can be applied in thecompositions and methods of the present invention may be any nucleicacid sequence that has been found by any source to be a validateddownregulator of the gene or gene product.

In some embodiments, the invention features an isolated siRNA moleculeof at least 19 nucleotides, having at least one strand that issubstantially complementary to at least ten but no more than thirtyconsecutive nucleotides of a nucleic acid that encodes a gene, and thatreduces the expression of a gene or gene product. In one embodiments ofthe present disclosure, the siRNA molecule has at least one strand thatis substantially complementary to at least ten but no more than thirtyconsecutive nucleotides of the mRNA that encodes a gene or a geneproduct.

In one embodiments, the siRNA molecule is at least 75, 80, 85, or 90%homologous, particularly at least 95%, 99%, or 100% similar oridentical, or any percentages in between the foregoing (e.g., theinvention contemplates 75% and greater, 80% and greater, 85% andgreater, and so on, and said ranges are intended to include all wholenumbers in between), to at least 10 contiguous nucleotides of any of thenucleic acid sequences encoding a target therapeutic protein.

The siRNA may also comprise an alteration of one or more nucleotides.Such alterations can include the addition of non-nucleotide material,such as to the end(s) of the 19 to 25 nucleotide RNA or internally (atone or more nucleotides of the RNA). In certain aspects, the RNAmolecule contains a 3′-hydroxyl group. Nucleotides in the RNA moleculesof the present invention can also comprise non-standard nucleotides,including non-naturally occurring nucleotides or deoxyribonucleotides.The double-stranded oligonucleotide may contain a modified backbone, forexample, phosphorothioate, phosphorodithioate, or other modifiedbackbones known in the art, or may contain non-natural internucleosidelinkages. Additional modifications of siRNAs (e.g., 2′-O-methylribonucleotides, 2′-deoxy-2′-fluoro ribonucleotides, “universal base”nucleotides, 5-C-methyl nucleotides, one or more phosphorothioateinternucleotide linkages, and inverted deoxyabasic residueincorporation) can be found in U.S. Publication 2004/0019001 and U.S.Pat. No. 6,673,611 (each of which is incorporated by reference in itsentirety). Collectively, all such altered nucleic acids or RNAsdescribed above are referred to as modified siRNAs.

In one embodiment, siRNA is capable of decreasing the expression of aparticular genetic product by at least 10%, at least 20%, at least 30%,or at least 40%, at least 50%, at least 60%, or at least 70%, at least75%, at least 80%, at least 90%, at least 95% or more or any ranges inbetween the foregoing.

3. CRISPR/CAS

CRISPRs (clustered regularly interspaced short palindromic repeats) areDNA loci containing short repetitions of base sequences. Each repetitionis followed by short segments of “spacer DNA” from previous exposures toa virus. CRISPRs are found in approximately 40% of sequenced eubacteriagenomes and 90% of sequenced archaea. CRISPRs are often associated withcas genes that code for proteins related to CRISPRs. The CRISPR/Cassystem is a prokaryotic immune system that confers resistance to foreigngenetic elements such as plasmids and phages and provides a form ofacquired immunity. CRISPR spacers recognize and silence these exogenousgenetic elements like RNAi in eukaryotic organisms.

Repeats were first described in 1987 for the bacterium Escherichia coli.In 2000, similar clustered repeats were identified in additionalbacteria and archaea and were termed Short Regularly Spaced Repeats(SRSR). SRSR were renamed CRISPR in 2002. A set of genes, some encodingputative nuclease or helicase proteins, were found to be associated withCRISPR repeats (the cas, or CRISPR-associated genes).

In 2005, three independent researchers showed that CRISPR spacers showedhomology to several phage DNA and extrachromosomal DNA such as plasmids.This was an indication that the CRISPR/cas system could have a role inadaptive immunity in bacteria. Koonin and colleagues proposed thatspacers serve as a template for RNA molecules, analogously to eukaryoticcells that use a system called RNA interference.

In 2007 Barrangou, Horvath (food industry scientists at Danisco) andothers showed that they could alter the resistance of Streptococcusthermophilus to phage attack with spacer DNA. Doudna and Charpentier hadindependently been exploring CRISPR-associated proteins to learn howbacteria deploy spacers in their immune defenses. They jointly studied asimpler CRISPR system that relies on a protein called Cas9. They foundthat bacteria respond to an invading phage by transcribing spacers andpalindromic DNA into a long RNA molecule that the cell then usestracrRNA and Cas9 to cut it into pieces called crRNAs.

CRISPR was first shown to work as a genome engineering/editing tool inhuman cell culture by 2012 It has since been used in a wide range oforganisms including bakers yeast (S. cerevisiae), zebra fish, nematodes(C. elegans), plants, mice, and several other organisms. AdditionallyCRISPR has been modified to make programmable transcription factors thatallow scientists to target and activate or silence specific genes.Libraries of tens of thousands of guide RNAs are now available.

The first evidence that CRISPR can reverse disease symptoms in livinganimals was demonstrated in March 2014, when MIT researchers cured miceof a rare liver disorder. Since 2012, the CRISPR/Cas system has beenused for gene editing (silencing, enhancing or changing specific genes)that even works in eukaryotes like mice and primates. By inserting aplasmid containing cas genes and specifically designed CRISPRs, anorganism's genome can be cut at any desired location.

CRISPR repeats range in size from 24 to 48 base pairs. They usually showsome dyad symmetry, implying the formation of a secondary structure suchas a hairpin, but are not truly palindromic. Repeats are separated byspacers of similar length. Some CRISPR spacer sequences exactly matchsequences from plasmids and phages, although some spacers match theprokaryote's genome (self-targeting spacers). New spacers can be addedrapidly in response to phage infection.

CRISPR-associated (cas) genes are often associated with CRISPRrepeat-spacer arrays. As of 2013, more than forty different Cas proteinfamilies had been described. Of these protein families, Cas1 appears tobe ubiquitous among different CRISPR/Cas systems. Particularcombinations of cas genes and repeat structures have been used to define8 CRISPR subtypes (Ecoli, Ypest, Nmeni, Dvulg, Tneap, Hmari, Apern, andMtube), some of which are associated with an additional gene moduleencoding repeat-associated mysterious proteins (RAMPs). More than oneCRISPR subtype may occur in a single genome. The sporadic distributionof the CRISPR/Cas subtypes suggests that the system is subject tohorizontal gene transfer during microbial evolution.

Exogenous DNA is apparently processed by proteins encoded by Cas genesinto small elements (˜30 base pairs in length), which are then somehowinserted into the CRISPR locus near the leader sequence. RNAs from theCRISPR loci are constitutively expressed and are processed by Casproteins to small RNAs composed of individual, exogenously-derivedsequence elements with a flanking repeat sequence. The RNAs guide otherCas proteins to silence exogenous genetic elements at the RNA or DNAlevel. Evidence suggests functional diversity among CRISPR subtypes. TheCse (Cas subtype Ecoli) proteins (called CasA-E in E. coli) form afunctional complex, Cascade, that processes CRISPR RNA transcripts intospacer-repeat units that Cascade retains. In other prokaryotes, Cas6processes the CRISPR transcripts. Interestingly, CRISPR-based phageinactivation in E. coli requires Cascade and Cas3, but not Cas1 andCas2. The Cmr (Cas RAMP module) proteins found in Pyrococcus furiosusand other prokaryotes form a functional complex with small CRISPR RNAsthat recognizes and cleaves complementary target RNAs. RNA-guided CRISPRenzymes are classified as type V restriction enzymes.

See also U.S. Patent Publication 2014/0068797, which is incorporated byreference in its entirety.

i. Cas9

Cas9 is a nuclease, an enzyme specialized for cutting DNA, with twoactive cutting sites, one for each strand of the double helix. It hasbeen demonstrated that one could disable one or both sites whilepreserving Cas9's ability to home located its target DNA. Jinek combinedtracrRNA and spacer RNA into a “single-guide RNA” molecule that, mixedwith Cas9, could find and cut the correct DNA targets. Jinek et al.proposed that such synthetic guide RNAs might be able to be used forgene editing.

Cas9 proteins are highly enriched in pathogenic and commensal bacteria.CRISPR/Cas-mediated gene regulation may contribute to the regulation ofendogenous bacterial genes, particularly during bacterial interactionwith eukaryotic hosts. For example, Cas protein Cas9 of Francisellanovicida uses a unique, small, CRISPR/Cas-associated RNA (scaRNA) torepress an endogenous transcript encoding a bacterial lipoprotein thatis critical for F. novicida to dampen host response and promotevirulence.

ii. gRNA or sgRNA

As an RNA guided protein, Cas9 requires a short RNA to direct therecognition of DNA targets (Mali et al., 2013a). Though Cas9preferentially interrogates DNA sequences containing a PAM sequence NGGit can bind here without a protospacer target. However, the Cas9-gRNAcomplex requires a close match to the gRNA to create a double strandbreak (Cho et al., 2013; Hsu et al., 2013). CRISPR sequences in bacteriaare expressed in multiple RNAs and then processed to create guidestrands for RNA (Bikard et al., 2013). Because Eukaryotic systems lacksome of the proteins required to process CRISPR RNAs the syntheticconstruct gRNA was created to combine the essential pieces of RNA forCas9 targeting into a single RNA expressed with the RNA polymerase typeIII promoter U6 (Mali et al., 2013a, b). Synthetic gRNAs are slightlyover 100 bp at the minimum length and contain a portion which is targetsthe 20 protospacer nucleotides immediately preceding the PAM sequenceNGG; gRNAs do not contain a PAM sequence.

4. Modified Nucleobases

In some embodiments, the nucleic acids of the present disclosurecomprise one or more modified nucleosides comprising a modified sugarmoiety. Such compounds comprising one or more sugar-modified nucleosidesmay have desirable properties, such as enhanced nuclease stability orincreased binding affinity with a target nucleic acid relative to anoligonucleotide comprising only nucleosides comprising naturallyoccurring sugar moieties. In some embodiments, modified sugar moietiesare substituted sugar moieties. In some embodiments, modified sugarmoieties are sugar surrogates. Such sugar surrogates may comprise one ormore substitutions corresponding to those of substituted sugar moieties.

In some embodiments, modified sugar moieties are substituted sugarmoieties comprising one or more non-bridging sugar substituent,including but not limited to substituents at the 2′ and/or 5′ positions.Examples of sugar substituents suitable for the 2′-position, include,but are not limited to: 2′-F, 2′-OCH₃ (“OMe” or “O-methyl”), and2′-O(CH₂)₂OCH₃ (“MOE”). In certain embodiments, sugar substituents atthe 2′ position is selected from allyl, amino, azido, thio, O-allyl,O—C₁-C₁₀ alkyl, O—C₁-C₁₀ substituted alkyl; OCF₃, O(CH₂)₂SCH₃,O(CH₂)₂—O—N(Rm)(Rn), and O—CH₂—C(═O)—N(Rm)(Rn), where each Rm and Rn is,independently, H or substituted or unsubstituted C₁-C₁₀ alkyl. Examplesof sugar substituents at the 5′-position, include, but are not limitedto: 5′-methyl (R or S); 5′-vinyl, and 5′-methoxy. In some embodiments,substituted sugars comprise more than one non-bridging sugarsubstituent, for example, T-F-5′-methyl sugar moieties (see, e.g., PCTInternational Application WO 2008/101157, for additional 5′,2′-bissubstituted sugar moieties and nucleosides).

Nucleosides comprising 2′-substituted sugar moieties are referred to as2′-substituted nucleosides. In some embodiments, a 2′-substitutednucleoside comprises a 2′-substituent group selected from halo, allyl,amino, azido, SH, CN, OCN, CF₃, OCF₃, O, S, or N(R_(m))-alkyl; O, S, orN(R_(m))-alkenyl; O, S or N(R_(m))-alkynyl; O-alkylenyl-O-alkyl,alkynyl, alkaryl, aralkyl, O-alkaryl, O-aralkyl, O(CH₂)₂SCH₃,O(CH₂)₂—O—N(R_(m))(R_(n)) or O—CH₂—C(═O)—N(R_(m))(R_(n)), where eachR_(m) and R is, independently, H, an amino protecting group orsubstituted or unsubstituted C₁-C₁₀ alkyl. These 2′-substituent groupscan be further substituted with one or more substituent groupsindependently selected from hydroxyl, amino, alkoxy, carboxy, benzyl,phenyl, nitro (NO₂), thiol, thioalkoxy (S-alkyl), halogen, alkyl, aryl,alkenyl and alkynyl.

In some embodiments, a 2′-substituted nucleoside comprises a2′-substituent group selected from F, NH₂, N₃, OCF₃, O—CH₃, O(CH₂)₃NH₂,CH₂—CH═CH₂, O—CH₂—CH═CH₂, OCH₂CH₂OCH₃, O(CH₂)₂SCH₃,O—(CH₂)₂—O—N(R_(m))(R_(n)), O(CH₂)₂O(CH₂)₂N(CH₃)₂, and N-substitutedacetamide (O—CH₂—C(═O)—N(R_(m))(R_(n)) where each R_(m) and R. is,independently, H, an amino protecting group or substituted orunsubstituted C₁-C₁₀ alkyl.

In some embodiments, a 2′-substituted nucleoside comprises a sugarmoiety comprising a 2′-substituent group selected from F, OCF₃, O—CH₃,OCH₂CH₂OCH₃, O(CH₂)₂SCH₃, O(CH₂)₂—O—N(CH₃)₂, —O(CH₂)₂O(CH₂)₂N(CH₃)₂, andO—CH₂—C(═O)—N(H)CH₃.

In some embodiments, a 2′-substituted nucleoside comprises a sugarmoiety comprising a 2′-substituent group selected from F, O—CH₃, andOCH₂CH₂OCH₃.

Certain modified sugar moieties comprise a bridging sugar substituentthat forms a second ring resulting in a bicyclic sugar moiety. In somesuch embodiments, the bicyclic sugar moiety comprises a bridge betweenthe 4′ and the 2′ furanose ring atoms. Examples of such 4′ to 2′ sugarsubstituents, include, but are not limited to: —[C(R_(a))(R_(b))]_(n)—,—[C(R_(a))(R_(b))]_(n)—O—, —C(R_(a)R_(b))—N(R)—O— or,—C(R_(a)R_(b))—O—N(R)—; 4′-CH₂-2′, 4′—(CH₂)₂-2′, 4′—(CH₂)—O-2′ (LNA);4′-(CH₂)—S-2′; 4′—(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ (cEt) and4′-CH(CH₂OCH₃)—O-2′, and analogs thereof (see, e.g., U.S. Pat. No.7,399,845); 4′-C(CH₃)(CH₃)—O-2′ and analogs thereof, (see, e.g., WO2009/006478); 4′-CH₂—N(OCH₃)-2′ and analogs thereof (see, e.g.,WO2008/150729); 4′-CH₂—O—N(CH₃)-2′ (see, e.g., US2004/0171570, publishedSep. 2, 2004); 4′-CH₂—O—N(R)-2′, and 4′-CH₂—N(R)—O-2′-, wherein each Ris, independently, H, a protecting group, or C₁-C₁₂ alkyl;4′-CH₂—N(R)—O-2′, wherein R is H, C₁-C₁₂ alkyl, or a protecting group(see, U.S. Pat. No. 7,427,672); 4′-CH₂—C(H)(CH₃)-2′ (see, e.g.,Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and4′-CH₂—C(═CH₂)-2′ and analogs thereof (see, PCT InternationalApplication WO 2008/154401).

In some embodiments, such 4′ to 2′ bridges independently comprise from 1to 4 linked groups independently selected from —[C(R_(a))(R_(b))]_(n)—,—C(R_(a))═C(R_(b))—, —C(R_(a))═N—, —C(═NR_(a))—, —C(═O)—, —C(═S)—, —O—,—Si(R_(a))₂—, —S(═O)_(x)—, and —N(R_(a))—; wherein:

-   -   x is 0, 1, or 2;    -   n is 1, 2, 3, or 4;    -   each R_(a) and R_(b) is, independently, H, a protecting group,        hydroxyl, C₁-C₁₂ alkyl, substituted C₁-C₁₂ alkyl, C₂-C₁₂        alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂ alkynyl, substituted        C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted C₅-C₂₀ aryl,        heterocycle radical, substituted heterocycle radical,        heteroaryl, substituted heteroaryl, C₅-C₇ alicyclic radical,        substituted C₅-C₇ alicyclic radical, halogen, OJ₁, NJ₁J₂, SJ₁,        N₃, COOJ₁, acyl (C(═O)—H), substituted acyl, CN, sulfonyl        (S(═O)₂-J₁), or sulfoxyl (S(═O)-J₁); and    -   each J₁ and J₂ is, independently, H, C₁-C₁₂ alkyl, substituted        C₁-C₁₂ alkyl, C₂-C₁₂ alkenyl, substituted C₂-C₁₂ alkenyl, C₂-C₁₂        alkynyl, substituted C₂-C₁₂ alkynyl, C₅-C₂₀ aryl, substituted        C₅-C₂₀ aryl, acyl (C(═O)—H), substituted acyl, a heterocycle        radical, a substituted heterocycle radical, C₁-C₁₂ aminoalkyl,        substituted C₁-C₁₂ aminoalkyl, or a protecting group.

Nucleosides comprising bicyclic sugar moieties are referred to asbicyclic nucleosides or BNAs. Bicyclic nucleosides include, but are notlimited to, (A) α-L-Methyleneoxy (4′-CH₂—O-2′) BNA, (B) β-D-Methyleneoxy(4′-CH₂—O-2′) BNA (also referred to as locked nucleic acid or LNA), (C)Ethyleneoxy (4′-(CH₂)₂—O-2′) BNA, (D) Aminooxy (4′-CH₂—O—N(R)-2′) BNA,(E) Oxyamino (4′-CH₂—N(R)—O-2′) BNA, (F) Methyl(methyleneoxy)(4′-CH(CH₃)—O-2′) BNA (also referred to as constrained ethyl or cEt),(G) methylene-thio (4′-CH₂—S-2′) BNA, (H) methylene-amino(4′-CH₂—N(R)-2′) BNA, (I) methyl carbocyclic (4′-CH₂—CH(CH₃)-2′) BNA,(J) propylene carbocyclic (4′-(CH₂)₃-2′) BNA, and (K)Methoxy(ethyleneoxy) (4′-CH(CH₂OMe)-O-2′) BNA (also referred to asconstrained MOE or cMOE).

Additional bicyclic sugar moieties are known in the art, for example:Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al.,Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad.Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem.Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63,10035-10039; Srivastava et al., J. Am. Chem. Soc., 129(26) 8362-8379(Jul. 4, 2007); Elayadi et al., Curr. Opinion Invens. Drugs, 2001, 2,5561; Braasch et al., Chem. Biol., 2001, 8, 1-7; Orum et al., Curr.Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos. 7,053,207,6,268,490, 6,770,748, 6,794,499, 7,034,133, 6,525,191, 6,670,461, and7,399,845; WO 2004/106356, WO 1994/14226, WO 2005/021570, and WO2007/134181; U.S. Patent Publication Nos. US 2004/0171570, US2007/0287831, and US 2008/0039618; U.S. Ser. Nos. 12/129,154,60/989,574, 61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787,and 61/099,844; and PCT International Applications Nos.PCT/US2008/064591, PCT/US2008/066154, and PCT/US2008/068922.

In some embodiments, bicyclic sugar moieties and nucleosidesincorporating such bicyclic sugar moieties are further defined byisomeric configuration. For example, a nucleoside comprising a 4′-2′methylene-oxy bridge, may be in the .alpha.-L configuration or in the.beta.-D configuration. Previously, α-L-methyleneoxy (4′-CH₂—O-2′)bicyclic nucleosides have been incorporated into antisenseoligonucleotides that showed antisense activity (Frieden et al., NucleicAcids Research, 2003, 21, 6365-6372).

In some embodiments, substituted sugar moieties comprise one or morenon-bridging sugar substituent and one or more bridging sugarsubstituent (e.g., 5′-substituted and 4′-2′ bridged sugars; PCTInternational Application WO 2007/134181, wherein LNA is substitutedwith, for example, a 5′-methyl or a 5′-vinyl group).

In some embodiments, modified sugar moieties are sugar surrogates. Insome such embodiments, the oxygen atom of the naturally occurring sugaris substituted, e.g., with a sulfer, carbon or nitrogen atom. In somesuch embodiments, such modified sugar moiety also comprises bridgingand/or non-bridging substituents as described above. For example,certain sugar surrogates comprise a 4′-sulfur atom and a substitution atthe 2′-position (see, e.g., published U.S. Patent Application US2005/0130923) and/or the 5′ position. By way of additional example,carbocyclic bicyclic nucleosides having a 4′-2′ bridge have beendescribed (see, e.g., Freier et al., Nucleic Acids Research, 1997,25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71,7731-7740).

In some embodiments, sugar surrogates comprise rings having other than5-atoms. For example, in some embodiments, a sugar surrogate comprises asix-membered tetrahydropyran. Such tetrahydropyrans may be furthermodified or substituted. Nucleosides comprising such modifiedtetrahydropyrans include, but are not limited to, hexitol nucleic acid(HNA), anitol nucleic acid (ANA), manitol nucleic acid (MNA) (seeLeumann, C J. Bioorg. & Med. Chem. (2002) 10:841-854), and fluoro HNA(F-HNA).

In some embodiments, the modified THP nucleosides of Formula VII areprovided wherein q₁, q₂, q₃, q₄, q₅, q₆ and q₇ are each H. In certainembodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ is other thanH. In some embodiments, at least one of q₁, q₂, q₃, q₄, q₅, q₆ and q₇ ismethyl. In some embodiments, THP nucleosides of Formula VII are providedwherein one of R₁ and R₂ is F. In certain embodiments, R₁ is fluoro andR₂ is H, R₁ is methoxy and R₂ is H, and R₁ is methoxyethoxy and R₂ is H.

Many other bicyclo and tricyclo sugar surrogate ring systems are alsoknown in the art that can be used to modify nucleosides forincorporation into antisense compounds (see, e.g., review article:Leumann, J. C, Bioorganic & Medicinal Chemistry, 2002, 10, 841-854).

Combinations of modifications are also provided without limitation, suchas 2′-F-5′-methyl substituted nucleosides (see PCT InternationalApplication WO 2008/101157 for other disclosed 5′,2′-bis substitutednucleosides) and replacement of the ribosyl ring oxygen atom with S andfurther substitution at the 2′-position (see U.S. Patent Publication US2005/0130923) or alternatively 5′-substitution of a bicyclic nucleicacid (see PCT International Application WO 2007/134181 wherein a4′-CH₂—O-2′ bicyclic nucleoside is further substituted at the 5′position with a 5′-methyl or a 5′-vinyl group). The synthesis andpreparation of carbocyclic bicyclic nucleosides along with theiroligomerization and biochemical studies have also been described (see,e.g., Srivastava et al., 2007).

In some embodiments, the present invention provides oligonucleotidescomprising modified nucleosides. Those modified nucleotides may includemodified sugars, modified nucleobases, and/or modified linkages. Thespecific modifications are selected such that the resultingoligonucleotides possess desirable characteristics. In some embodiments,oligonucleotides comprise one or more RNA-like nucleosides. In someembodiments, oligonucleotides comprise one or more DNA-like nucleotides.

In some embodiments, nucleosides of the present invention comprise oneor more unmodified nucleobases. In certain embodiments, nucleosides ofthe present invention comprise one or more modified nucleobases.

In some embodiments, modified nucleobases are selected from: universalbases, hydrophobic bases, promiscuous bases, size-expanded bases, andfluorinated bases as defined herein. 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil; 5-propynylcytosine;5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynylCH₃) uracil and cytosine and other alkynyl derivatives of pyrimidinebases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 2-F-adenine, 2-aminoadenine,8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine,3-deazaguanine and 3-deazaadenine, universal bases, hydrophobic bases,promiscuous bases, size-expanded bases, and fluorinated bases as definedherein. Further modified nucleobases include tricyclic pyrimidines suchas phenoxazine cytidine([5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.,9-(2-aminoethoxy)-H-pyrimido[5,4-13][1,4]benzoxazin-2(3H)-one),carbazole cytidine (²H-pyrimido[4,5-b]indol-2-one), pyridoindolecytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modifiednucleobases may also include those in which the purine or pyrimidinebase is replaced with other heterocycles, for example 7-deazaadenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz,J. I., Ed., John Wiley & Sons, 1990, 858-859; those disclosed byEnglisch et al., 1991; and those disclosed by Sanghvi, Y. S., 1993.

Representative United States Patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include without limitation, U.S. Pat. Nos.3,687,808; 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985;5,681,941; 5,750,692; 5,763,588; 5,830,653 and 6,005,096, each of whichis herein incorporated by reference in its entirety.

In some embodiments, the present invention provides oligonucleotidescomprising linked nucleosides. In such embodiments, nucleosides may belinked together using any internucleoside linkage. The two main classesof internucleoside linking groups are defined by the presence or absenceof a phosphorus atom. Representative phosphorus containinginternucleoside linkages include, but are not limited to,phosphodiesters (P═O), phosphotriesters, methylphosphonates,phosphoramidate, and phosphorothioates (P═S). Representativenon-phosphorus containing internucleoside linking groups include, butare not limited to, methylenemethylimino (—CH₂—N(CH₃)—O—CH₂—),thiodiester (—O—C(O)—S—), thionocarbamate (—O—C(O)(NH)—S—); siloxane(—O—Si(H)₂—O—); and N,N′-dimethylhydrazine (—CH₂—N(CH₃)—N(CH₃)—).Modified linkages, compared to natural phosphodiester linkages, can beused to alter, typically increase, nuclease resistance of theoligonucleotide. In some embodiments, internucleoside linkages having achiral atom can be prepared as a racemic mixture, or as separateenantiomers. Representative chiral linkages include, but are not limitedto, alkylphosphonates and phosphorothioates. Methods of preparation ofphosphorous-containing and non-phosphorous-containing internucleosidelinkages are well known to those skilled in the art.

The oligonucleotides described herein contain one or more asymmetriccenters and thus give rise to enantiomers, diastereomers, and otherstereoisomeric configurations that may be defined, in terms of absolutestereochemistry, as (R) or (S), α or β such as for sugar anomers, or as(D) or (L) such as for amino acids etc. Included in the antisensecompounds provided herein are all such possible isomers, as well astheir racemic and optically pure forms.

Neutral internucleoside linkages include without limitation,phosphotriesters, methylphosphonates, MMI (3′-CH₂—N(CH₃)—O-5′), amide-3(3′-CH₂—C(═O)—N(H)-5′), amide-4 (3′-CH₂—N(H)—C(═O)-5′), formacetal(3′-O—CH₂—O-5′), and thioformacetal (3′-S—CH₂—O-5′). Further neutralinternucleoside linkages include nonionic linkages comprising siloxane(dialkylsiloxane), carboxylate ester, carboxamide, sulfide, sulfonateester and amides (See for example: Carbohydrate Modifications inAntisense Research; Y. S. Sanghvi and P. D. Cook, Eds., ACS SymposiumSeries 580; Chapters 3 and 4, 40-65). Further neutral internucleosidelinkages include nonionic linkages comprising mixed N, O, S and CH₂component parts.

Additional modifications may also be made at other positions on theoligonucleotide, particularly the 3′ position of the sugar on the 3′terminal nucleotide and the 5′ position of 5′ terminal nucleotide. Forexample, one additional modification of the ligand conjugatedoligonucleotides of the present invention involves chemically linking tothe oligonucleotide one or more additional non-ligand moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. Such moieties include but are not limitedto lipid moieties such as a cholesterol moiety (Letsinger et al., 1989),cholic acid (Manoharan et al., 1994), a thioether, e.g.,hexyl-5-tritylthiol (Manoharan et al., 1992; Manoharan et al., 1993), athiocholesterol (Oberhauser et al., 1992), an aliphatic chain, e.g.,dodecandiol or undecyl residues (Saison-Behmoaras et al., 1991; Kabanovet al., 1990; Svinarchuk et al., 1993), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., 1995;Shea et al., 1990), a polyamine or a polyethylene glycol chain(Manoharan et al., 1995), or adamantane acetic acid (Manoharan et al.,1995), a palmityl moiety (Mishra et al., 1995), or an octadecylamineorhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., 1996).

Representative United States patents that teach the preparation of sucholigonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of whichis herein incorporated by reference.

E. KITS

The present disclosure also provides kits. Any of the componentsdisclosed herein may be combined in the form of a kit. In someembodiments, the kits comprise a dendrimer or a composition as describedabove or in the claims.

The kits will generally include at least one vial, test tube, flask,bottle, syringe or other container, into which a component may beplaced, and preferably, suitably aliquoted. Where there is more than onecomponent in the kit, the kit also will generally contain a second,third or other additional containers into which the additionalcomponents may be separately placed. However, various combinations ofcomponents may be comprised in a container. In some embodiments, all ofthe nucleic acid delivery components are combined in a single container.In other embodiments, some or all of the dendrimer delivery componentswith the instant polymers are provided in separate containers.

The kits of the present invention also will typically include packagingfor containing the various containers in close confinement forcommercial sale. Such packaging may include cardboard or injection orblow molded plastic packaging into which the desired containers areretained. A kit may also include instructions for employing the kitcomponents. Instructions may include variations that can be implemented.

F. EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1: Materials and Instrumentation

1. Materials for Chemical Synthesis

All amines, thiols, and otherwise unspecified chemicals were purchasedfrom Sigma-Aldrich. 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)was purchased from Avanti Lipids. Lipid PEG2000 was chemicallysynthesized, as described below. C12-200 was synthesized following thereported procedure (Love et al., 2010). All organic solvents werepurchased from Fisher Scientific and purified with a solventpurification system (Innovative Technology).

2. Nucleic Acids and Other Materials for In Vitro and In VivoExperiments

All siRNAs were purchased from Sigma-Aldrich. Let-7g miRNA mimic and itscontrol mimic were purchased from Ambion by Life Technologies.Dulbecco's Modified Eagle Media (DMEM) and fetal bovine serum (FBS) werepurchased from Sigma-Aldrich. OptiMEM, DAPI, and Alexa Fluor 488phalloidin were purchased from Life Technologies. ONE-Glo+Tox waspurchased from Promega. Biophen FVII was purchased from AniaraCorporation.

The sequence for the sense and antisense strands of siRNAs were asfollows:

siLuc siRNA against Luciferase). dT are DNA bases. All others are RNAbases.

sense: (SEQ ID NO: 3) 5′-GAUUAUGUCCGGUUAUGUA[dT][dT]-3′ antisense:(SEQ ID NO: 4) 3′-UACAUAACCGGACAUAAUC[dT][dT]-5′

siFVII (siRNA against FVII). 2′-Fluoro modified nucleotides are lowercase.

sense: (SEQ ID NO: 1) 5′-GGAucAucucAAGucuuAc[dT][dT]-3′ antisense:(SEQ ID NO: 2) 3′-GuAAGAcuuGAGAuGAucc[dT][dT]-5′

siCTR (siRNA as control)

sense: (SEQ ID NO: 5) 5′-GCGCGAUAGCGCGAAUAUA[dT][dT]-3′ antisense:(SEQ ID NO: 6) 3′-UAUAUUCGCGCUAUCGCGC[dT][dT]-5′

Sigma-Aldrich MISSION siRNA Universal Negative Control #1 (catalognumber: SIC001) was used as a non-targeted siRNA in control experiments.2′ OMe modified control siRNAs (Sigma-Aldrich, proprietarymodifications) were used in in vivo studies to reduce immunestimulation.

Cy5.5-labeled siRNA (siRNA for imaging)

sense: (SEQ ID NO: 3) 5′-Cy5.5-GAUUAUGUCCGGUUAUGUA[dT][dT]-3′ antisense:(SEQ ID NO: 4) 3′-UACAUAACCGGACAUAAUC[dT][dT]-5′

Let-7g miRNA mimic

Ambion (Life Technologies) mirVana miRNA mimic (catalog number: 4464070,product ID: MC11758, name: has-let-7g). Exact sequence and modificationsnot disclosed by Ambion. Mimics mature human Let-7g.

Negative control (CTR) miRNA mimic

Ambion (Life Technologies) mirVana miRNA Mimic, Negative Control #1(catalog number: 4464061). Exact sequence and modifications notdisclosed by Ambion.

3. Robotic Automation

Nanoparticle (NP) formulations and in vitro screening were performed ona Tecan Freedom EVO 200 fluid handling robot equipped with an 8-channelliquid handling arm (LiHa), multi-channel arm with 96-channel head(MCA), robotic manipulator arm (RoMa), and an integrated InfiniTe F/M200Pro microplate reader (Tecan). Two integrated custom heating andstirring chemical reaction stations (V&P Scientific 710E-3HM SeriesTumble Stirrers) provided reaction and mixing support. All operationswere programmed in EVOware Standard software (Tecan).

4. Synthetic Characterization

¹H and ¹³C NMR were performed on a Varian 500 MHz spectrometer. MS wasperformed on a Voyager DE-Pro MALDI TOF. Flash chromatography wasperformed on a Teledyne Isco CombiFlash Rf-200i chromatography systemequipped with UV-vis and evaporative light scattering detectors (ELSD).Particle sizes and zeta potentials were measured by Dynamic LightScattering (DLS) using a Malvern Zetasizer Nano ZS (He—Ne laser, λ=632nm).

5. Nanoparticle Formulation for In Vivo Studies

Formulated dendrimer nanoparticles for in vivo studies were preparedusing a microfluidic mixing instrument with herringbone rapid mixingfeatures (Precision Nanosystems NanoAssemblr). Ethanol solutions ofdendrimers, DSPC, cholesterol, and lipid PEG2000 were rapidly combinedwith acidic solutions of siRNA as described below. The typical ratio ofaqueous:EtOH was 3:1 (volume) and the typical flow rate was 12mL/minute.

6. Automated, In Vitro Delivery Screening of Modular DegradableDendrimers

Nanoparticle (NP) formulations and in vitro screening were performed ona Tecan Freedom EVO 200 fluid handling robot equipped with an 8-channelliquid handling arm (LiHa), multi-channel arm with 96-channel head(MCA), robotic manipulator arm (RoMa), and an integrated InfiniTe F/M200Pro microplate reader (Tecan).

HeLa cells stably expressing firefly luciferase (HeLa-Luc) were derivedfrom HeLa cells (ATCC) by stable transfection of the luciferase geneusing lentiviral infection followed by clonal selection. HeLa-Luc cellswere seeded (10,000 cells/well) into each well of an opaque white96-well plate (Corning) and allowed to attach overnight in phenolred-free DMEM supplemented with 5% FBS. The media was replaced withfresh, FBS-containing media on the second day before starting thetransfection.

G1DD-siLuc nanoparticles were formulated with the aid of an automated,fluid-handling robot to accelerate the discovery process. All operationswere programmed in EVOware Standard software. First, dendrimer reactionsolutions were diluted from the original reaction concentration to 12.5mM in ethanol. Next, the dendrimer solutions were diluted a second timefrom 12.5 mM to 1 mM in ethanol using the LiHa arm. Then, 89.2 μL of alipid mixture in ethanol was added into a 96-well clear plate. The lipidmixture was composed of DSPC (0.0690 mM), cholesterol (0.2622 mM), andlipid PEG2000 (0.0138 mM) in ethanol. Subsequently, 30.8 μL of eachdendrimer (1 mM) was added to the lipid mixture in the 96-well plate viathe LiHa, followed by rapid mixing (15 times; 75 μL mixing volume; 250μL/second speed). The LiHa added and mixed 8 tips at once. To a secondclear 96-well plate, 50 μL of siLuc (20 ng/μL) in citrate buffer(pH=4.3) was added via the LiHa. 30 μL of the ethanol mixture(dendrimer, DSPC, cholesterol, lipid PEG2000) was then added to the 50μL siLuc solution, followed by rapid mixing (15 times; 75 μL mixingvolume; 250 μL/second speed) to form the dendrimer nanoparticles. Next,120 μL of sterile PBS (lx) was added and mixed using the LiHa to dilutethe NPs and increase the pH. Subsequently, the plates were re-formattedto allow for facile transfer to growing cells. Finally, 20 μL of the NPsolutions was added to culturing cells using sterile disposable tips viathe MCA96 head to avoid contamination. The cells ultimately received 100ng siLuc (33 nM). The mol ratio of dendrimer to siLuc was 100:1 duringthis screening phase. The final composition of the formulation wasG1DD:cholesterol:DSPC:lipid PEG2000: =50:38:10:2 (by mole). Cells wereincubated for 24 h at 37° C., 5% CO₂ and then firefly luciferaseactivity and viability was analyzed using One Glo+Tox assay kits(Promega).

7. Dendrimer-Small RNA Formulations for In Vivo Studies

Formulated dendrimer nanoparticles for in vivo studies were preparedusing a microfluidic mixing instrument with herringbone rapid mixingfeatures (Precision Nanosystems NanoAssemblr). Ethanol solutions ofdendrimer, DSPC, cholesterol, and lipid PEG2000 (molar ratio of50:38:10:2) were rapidly combined with acidic solutions of small RNA togive the final weight ratio of 25:1 (dendrimer:small RNA). The typicalratio of aqueous:EtOH was 3:1 (volume) and the typical flow rate was 12mL/minute. C12-200 LNPs were prepared according to the reportedprocedure (Love et al., 2010). Ethanol solutions of C12-200, DSPC,cholesterol, and lipid PEG2000 (molar ratio of 50:38.5:10:1.5) wererapidly combined with acidic solutions of small RNA to give the finalweight ratio of 7:1 (C12-200:small RNA). All formulated NPs werepurified by dialysis in sterile PBS with 3.5 kD cut-off and the size wasmeasured by Dynamic Light Scattering (DLS) prior to in vivo studies.When applicable, the encapsulation of small RNAs was measured withRibogreen binding assay (Invitrogen) by taking the small amount ofsolution and following its protocol.

8. Animal Studies

All experiments were approved by the Institutional Animal Care and UseCommittees of The University of Texas Southwestern Medical Center andwere consistent with local, state and federal regulations as applicable.Female C57BL/6 mice were purchased from Harlan Laboratories(Indianapolis, Ind.). Transgenic mice bearing MYC-driven liver tumorswere generated by crossing the TRE-MYC strain with LAP-tTA strain. Micebearing the LAP-tTA and TRE-MYC genotype were maintained on 1 mg/mL ofdox, and MYC was induced by withdrawing dox. Power analysis wasperformed to anticipate required number of animals to achievestatistical significance.

9. In Vivo Factor VII Silencing in Mice

For in vivo delivery screening, female C57BL/6 mice received tail veini.v. injections of PBS (negative control, n=3) or dendrimer NPscontaining non-targeting siRNA (siCTR, negative control, n=3) ordendrimer NPs containing anti-Factor VII siRNA (siFVII, n=3) diluted inPBS (200 μL or less in total volume). After 48 h, body-weight gain/losswas measured and mice were anaesthetized by isofluorane inhalation forblood sample collection by retro-orbital eye bleed. Serum was isolatedwith serum separation tubes (Becton Dickinson) and Factor VII proteinlevels were analyzed by a chromogenic assay (Biophen FVII, AniaraCorporation). A standard curve was constructed using samples fromPBS-injected mice and relative Factor VII expression was determined bycomparing treated groups to an untreated PBS control.

For the therapeutic study, FVII knockdown in transgenic mice wereverified with the above blood assay and by qPCR using harvested livertissues. To evaluate statistical significance, two-tailed Student'st-tests with the 95% confidence level were conducted.

10. Biodistribution

Female C57BL/6 mice or transgenic mice bearing liver tumors receivedtail vein i.v. injections with dendrimer NPs containing Cy5.5-siRNA at 1mg/kg of siRNA in 200 μL. At 24 h post injection, mice were euthanizedand organs were removed. The biodistribution was assessed by imagingwhole organs with an IVIS Lumina System (Caliper Life Sciences) with theCy5.5 filter setting.

For confocal imaging, the tissue was cryo-sectioned (7 μm) and fixedusing 4% paraformaldehyde at room temperature for 10 min. The slideswere washed three times with PBS and blocked for 30 min in PBS with 1%albumin. Sections were then incubated for 30 min with Alexa Fluor 488Phalloidin (1:200 dilution, Life Technologies) in PBS with 1% albumin.Slides were washed three times with 0.1% Tween 20 and mounted usingProLong Gold Antifade (Life Technologies). Sections were imaged using anLSM 700 point scanning confocal microscope (Zeiss) equipped with a 25×objective.

11. In Vivo Toxicity Evaluation and Let-7g Therapeutic Studies

Wild-type mice or transgenic mice bearing liver tumors were randomlydivided into different groups. Mice received tail vein i.v. injectionsof dendrimer NPs containing siCTR. Their body weight was monitoreddaily. For transgenic mice bearing liver tumors, multiple tail vaininjections were performed to simulate repeated dosing.

For Let-7g therapeutic studies, transgenic mice bearing liver tumorsreceived weekly tail vein i.v. injections of dendrimer NPs with Let-7gmimic or CTR mimic at a dosage of 1 mg/kg in 200 μL PBS from the age of26 to 61 days. Processing order randomization was used. No blinding wasdone. Their body weight, abdomen size, and survival were carefullymonitored. To evaluate statistical significance, two-tailed T tests withthe 95% confidence level or Mantel-Cox tests were conducted.

Example 2: Synthesis and Characterization of PEG Lipids and Dendrimers

1. Synthesis of Library Containing 1,512 First-Generation DegradableDendrimers (G1DDs)

G1DDs were synthesized through two sequential orthogonal reactions. Atfirst, amines with different initial branching centers (IBCs) wereseparately reacted with the acrylate group of 2-(acryloyloxy)ethylmethacrylate (AEMA) with the mole ratio of amine to AEMA equaling theIBC numbers (e.g. 2A amines: two equivalents of AEMA were added; 6Aamines: six equivalents of AEMA were added). Reactions were conductedwith the addition of 5 mol % butylated hydroxyltoluene (BHT) for 24hours at 50° C. Next, each first-step adduct was reacted separately witheach thiol at the mole ratio of thiol to adduct equaling the amine IBCnumbers (e.g. 2A amine first-step adduct: two equivalents of each thiolwas added; 6A amine first-step adduct: six equivalents of each thiol wasadded). Reactions were conducted with the addition of 5 mol %dimethylphenylphosphine (DMPP) catalyst for 48 hours at 60° C. The 1,512member library synthesis was accelerated by conducting reactions inglass vials and aluminum reaction blocks. Custom heating and stirringchemical reaction stations (V&P Scientific 710E-3HM Series TumbleStirrers) were employed.

Initial in vitro delivery screening experiments were conducted withcrude G1DDs. Follow-up studies to verify activity were performed usingpurified dendrimers.

All in vivo animal experiments were performed with purified G1DDs.Purified G1DDs were obtained by column flash chromatography on a neutralalumina column using a Teledyne Isco chromatography system with thegradient eluent of hexane and ethyl acetate.

2. Synthesis of Higher Generation Degradable Dendrimers (HGDDs)(1A2-G2-SC8 as an Example)

Higher generation degradable dendrimers were prepared according to theprevious method (Ma et al., 2009). 1A2-G1 was prepared directly after1A2 amine reacted with one equivalent of AEMA in the presence of 5 mol %BHT at 50° C. for 24 hours. 1A2-G1 (4.00 g, 11.7 mmol) was dissolved in10 mL DMSO. After addition of 2-aminoethanthiol (1.37 g, 17.5 mmol) intothe above solution, the reaction was stirred at room temperature for 30min. Then 300 mL dichloromethane was immediately added into the reactionsolution and was washed with cold brine water (50 mL×3) to remove extra2-aminoethanthiol. The organic phase was dried with magnesium sulfateand condensed via rotary evaporation to use directly for next step. AEMA(4.75 g, 25.8 mmol) and BHT (227 mg, 1.08 mmol) were added into theabove solution. The reaction was stirred at 50° C. and monitored by ¹HNMR. After the reaction was complete, the solution was repeatedly washedwith 20 mL hexane portions until no EAMA was found through TLC plateanalysis. The washed solution was dried in vacuum to yield a viscousliquid 1A3-G2 directly for the next step. 1A3-G2 was reacted byfollowing the above two-step synthetic procedure to give the viscousliquid 1A3-G3 directly for next the step. After 1A2-G3 (0.5 g, 0.3 mmol)was dissolved in 0.5 mL DMSO, 1-octanethiol (216 μL, 1.22 mmol) anddimethylphenylphosphine (DMPP) (8.6 μL, 0.061 mmol) was added. Thereaction was stirred at 60° C. for 48 hours and then purified by runninga neutral alumina column with the gradient eluent of hexane and ethylacetate. A light-yellow viscous liquid 1A2-G3-SC8 was obtained.

3. Synthesis of Lipid PEG2000

PEG₄₄-OH (80 g, 40 mmol) and pyridine (6.5 mL, 80 mmol) were dissolvedin 250 mL anhydrous DCM and cooled at 0° C. Methanesulfonyl chloride(15.5 mL, 200 mmol) in 50 mL DCM was added over 30 min and the mixturewas stirred overnight at room temperature. Another 100 mL DCM was addedand the organic phase washed with saturated NaHCO₃ solution (50 mL×3),and then brine (50 mL×3). The resulting solution was concentrated andthe residue was recrystallized in isopropanol and dried to yield a whitepowder PEG2000-Ms (74 g, 93%).

PEG2000-Ms (35.41 g, 17.7 mmol) was dissolved in 250 mL of DMF. Then,NaN₃ (12.4 g, 19.0 mmol) was added into the solution. The reaction wasstirred under nitrogen for 2 days at 50° C. After removal of DMF, theresidual was dissolved in 300 mL DCM and washed with brine (50 mL×3).After removal of solvents, the residual oil was dissolved in 50 mL ofmethanol and the product was precipitated three times with 300 mL ofdiethyl ether to give the desired compound (25.55 g, 72%) as a whitepowder PEG2000-N₃.

Propargylamine (0.50 g, 9.1 mmol), BHT (191 mg, 0.91 mmol), and EAMA(2.73 g, 18.2 mmol) were added into a 25 mL reaction vial. The mixturewas stirred for 48 hours at 50° C. The reaction was cooled down to givea colorless oil product T3-G1 without purification for use in the nextreaction.

4. Characterization of Select Dendrimers

¹H NMR (400 MHz, CDCl₃, δ): 4.38-4.19 (br, 28H, —OCH₂CH₂O—), 2.90-2.80(br, 7H, —C(O)CH(CH₃)CH₂S—), 2.75-2.71 (br, 14H, —NCH₂CH₂C(O)—),2.70-2.49 (br, 28H, —C(O)CH(CH₃)CH₂S—, —SCH₂—), 2.49-2.39 (br, 36H,—N(CH₃)₂, —NCH₂CH₂N(CH₂CH₂)₂NCH₂—, —CH₂N(CH₂-)₂), 1.57-1.48 (m, 8H,—SCH₂CH₂CH₂—), 1.37-1.28 (br, 8H, SCH₂CH₂CH₂—), 1.28-1.16 (br, 53H,—SCH₂CH₂(CH₂)₄CH₃, —CHC(CH₃)CH₂S—), 0.85 (t, J=7.1 Hz, 12H, —(CH₂)₄CH₃).¹³C NMR (400 MHz, CDCl₃, δ): 174.92, 172.03, 62.22, 62.17, 62.13, 62.07,49.06, 40.23, 40.14, 35.36, 32.68, 32.56, 31.76, 29.60, 29.14, 28.82,22.58, 16.85, 16.81, 14.04. MS (MALDI-TOF, m/z) Calc. forC₁₀₉H₁₉₆N₆O₂₈S₇: 2261.21, found: 2262.43.

¹H NMR (400 MHz, CDCl₃, δ): 4.34-4.21 (br, 16H, —OCH₂CH₂O—), 2.82-2.76(m, 4H, —SCH₂CH(CH₃)—), 2.73 (t, J=7.1 Hz, 8H, —C(O)CH₂CH₂N—), 2.70-2.64(m, 4H, —SCH₂CH(CH₃)—), 2.58-2.51 (m, 4H, —SCH₂CH(CH₃)—), 2.51-2.46 (m,8H, —CH₂CH₂S—), 2.45-2.40 (m, 18H, (—C(O)CH₂CH₂)₂NCH₂CH₂CH₂N(CH₂-)₂),2.35-2.26 (br, 4H, —CH₂CH₂N(CH₂-)₂), 1.65-1.58 (br, 4H, —NCH₂CH₂CH₂N—),1.57-1.49 (m, 8H, —SCH₂CH₂CH₂—), 1.37-1.28 (br, 8H, —SCH₂CH₂CH₂—),1.28-1.16 (br, 44H, —SCH₂CH₂(CH₂)₄CH₃, —CHC(CH₃)CH₂S—), 0.85 (t, J=7.0Hz, 12H, —(CH₂)₄CH₃). ¹³C NMR (400 MHz, CDCl₃, δ): 174.90, 172.18,62.18, 62.05, 49.05, 40.14, 35.37, 32.68, 32.40, 31.76, 29.60, 29.15,28.83, 22.60, 16.81, 14.08. MS (MALDI-TOF, m/z) Calc. forC₇₈H₁₄₄N₄O₁₆S₄: 1520.95, found: 1521.32.

¹H NMR (400 MHz, CDCl₃, δ): 4.32-4.21 (br, 16H, —OCH₂CH₂O—), 2.82-2.76(m, 4H, —SCH₂CH(CH₃)—), 2.73 (t, J=7.0 Hz, 8H, —C(O)CH₂CH₂N—), 2.69-2.62(m, 4H, —SCH₂CH(CH₃)—), 2.58-2.50 (m, 4H, —SCH₂CH(CH₃)—), 2.50-2.45 (m,8H, —CH₂CH₂S—), 2.45-2.38 (m, 12H, (—C(O)CH₂CH₂)₂NCH₂—), 2.34-2.24 (br,4H, —CH₂N(CH₃)CH₂—), 2.24-2.00 (br, 3H, —CH₂N(CH₃)CH₂—) 1.66-1.57 (br,4H, —NCH₂CH₂CH₂N—), 1.57-1.48 (m, 8H, —SCH₂CH₂CH₂—), 1.37-1.28 (br, 8H,—SCH₂CH₂CH₂—), 1.28-1.16 (br, 45H, —SCH₂CH₂(CH₂)₄CH₃, —CHC(CH₃)CH₂S—),0.85 (t, J=7.0 Hz, 12H, —(CH₂)₄CH₃). ¹³C NMR (400 MHz, CDCl₃, δ):174.90, 172.18, 62.18, 62.05, 49.00, 40.13, 35.36, 32.68, 32.35, 31.76,29.60, 29.15, 28.83, 22.60, 16.81, 14.04. MS (MALDI-TOF, m/z) Calc. forC₇₅H₁₃₉N₃O₁₆S₄: 1465.90, found: 1465.65.

¹H NMR (400 MHz, CDCl₃, δ): 4.34-4.20 (br, 20H, —OCH₂CH₂O—), 2.82-2.76(m, 5H, —SCH₂CH(CH₃)—), 2.75-2.70 (br, 10H, —C(O)CH₂CH₂N—), 2.69-2.62(m, 5H, —SCH₂CH(CH₃)—), 2.60-2.52 (m, 5H, —SCH₂CH(CH₃)—), 2.52-2.49 (m,10H, —CH₂CH₂S—), 2.49-2.45 (br, 16H, —NCH₂CH₂N—), 2.45-2.40 (br, 10H,—CH₂N—), 1.57-1.48 (br, 10H, —SCH₂CH₂CH₂—), 1.37-1.28 (br, 10H,—SCH₂CH₂CH₂—), 1.28-1.16 (br, 55H, —SCH₂CH₂(CH₂)₄CH₃, —CHC(CH₃)CH₂S—),0.87-0.79 (br, 15H, —(CH₂)₄CH₃). ¹³C NMR (400 MHz, CDCl₃, δ): 174.93,172.13, 62.28, 62.01, 49.04, 40.13, 35.36, 32.68, 32.35, 31.76, 29.60,29.15, 28.83, 22.59, 16.82, 14.05. MS (MALDI-TOF, m/z) Calc. forC₉₃H₁₇₃N₅O₂OS₅: 1840.13, found: 1841.37. 5A2-SC8 has also been preparedwith 6 arms (structure shown below).

¹H NMR (400 MHz, CDCl₃, δ): 4.32-4.21 (br, 20H, —OCH₂CH₂O—), 2.82-2.76(m, 5H, —SCH₂CH(CH₃)—), 2.76-2.70 (br, 10H, —C(O)CH₂CH₂N—), 2.69-2.62(m, 5H, —SCH₂CH(CH₃)—), 2.58-2.50 (m, 5H, —SCH₂CH(CH₃)—), 2.50-2.45 (m,10H, —CH₂CH₂S—), 2.45-2.20 (br, 20H, (—(CH₂)₂NCH₂—, —CH₂NHCH₂—),1.66-1.57 (br, 6H, —NCH₂CH₂CH₂N—), 1.57-1.48 (br, 10H, —SCH₂CH₂CH₂—),1.37-1.28 (br, 10H, —SCH₂CH₂CH₂—), 1.28-1.16 (br, 55H,—SCH₂CH₂(CH₂)₄CH₃, —CHC(CH₃)CH₂S—), 0.82-0.75 (br, 15H, —(CH₂)₄CH₃). ¹³CNMR (400 MHz, CDCl₃, δ): 174.98, 172.13, 62.28, 62.01, 49.04, 40.13,35.36, 32.68, 32.35, 31.76, 29.60, 29.15, 28.83, 22.61, 16.85, 14.14. MS(MALDI-TOF, m/z) Calc. for C₉₄H₁₇₄N₄O₂₀S₅: 1839.13, found: 1838.97.

¹H NMR (400 MHz, CDCl₃, δ): 4.33-4.20 (br, 24H, —OCH₂CH₂O—), 2.82-2.77(m, 6H, —SCH₂CH(CH₃)—), 2.77-2.71 (br, 12H, —C(O)CH₂CH₂N—), 2.68-2.62(m, 6H, —SCH₂CH(CH₃)—), 2.60-2.52 (m, 6H, —SCH₂CH(CH₃)—), 2.52-2.48 (br,12H, —CH₂CH₂S—), 2.48-2.46 (br, 12H, —NCH₂CH₂N—), 2.45-2.40 (br, 12H,(—CH₂)₂N—), 1.57-1.47 (br, 12H, —SCH₂CH₂CH₂—), 1.37-1.28 (br, 12H,—SCH₂CH₂CH₂—), 1.28-1.16 (br, 108H, —SCH₂CH₂(CH₂)₈CH₃, —CHC(CH₃)CH₂S—),0.87-0.80 (br, 18H, —(CH₂)₈CH₃). ¹³C NMR (400 MHz, CDCl₃, δ): 174.87,172.07, 62.16, 62.04, 49.48, 40.47, 40.11, 35.34, 32.69, 32.42, 31.86,29.61, 29.58, 29.57, 29.50, 29.29, 29.21, 28.85, 22.62, 16.81, 14.06. MS(MALDI-TOF, m/z) Calc. for C₁₃₂H₂₄₆N₄O₂₄S₆: 2463.65, found: 2464.52.

Example 3: Library Design and Synthesis of First Generation DegradableDendrimers (GIDDs)

Liver cancer is a challenging host for therapeutic intervention becausedrug-induced hepatotoxicity can exacerbate the underlying liver disease(Boyerinas et al., 2010). To achieve effective RNAi-mediated therapy, abalance of high potency and low toxicity of the carrier therefore has tobe maintained. This requires a versatile strategy to easily tune thedelivery carrier in terms of size, chemical structure, and ultimatephysical properties (FIG. 1A). In some embodiments, dendrimers weredesigned that exhibit one or more of the following characteristics:optimal, monodisperse materials for chemical and size manipulation (Wuet al., 2004; Carlmark et al., 2009; Killops et al., 2008; Ma et al.,2009; Franc and Kakkar, 2010). Orthogonal reactions were utilized tosequentially react with 2-(acryloyloxy)ethyl methacrylate (AEMA) todiversify first generation degradable dendrimers (G1DDs) through variousparameters: core (C), linkage or repeating unit (L), and periphery orterminating group (P) (FIG. 1B). In some embodiments, esters were chosenas a starting degradable linkage because polyesters are used inFDA-approved products with minimal toxicity. At each growth step, theester number increases, which provides an opportunity to identifydegradable dendrimers with balanced potency and toxicity.

Previous results show that these orthogonal reactions can constructpolyester dendrimers with a series of generations (Ma et al., 2009).However, before this strategy was utilized, it was verified that themethods are capable to yield diversified dendrimers using a variety ofchemically distinct amine and thiol compounds without purification. Toexamine the robustness of this chemistry, the most difficult startingmaterials, tris(2-aminoethyl)amine with six N—H bonds as initialbranching centers (IBCs) and tetradecylamine with a 14-carbon-lengthalkyl chain, were used to test structural limits of the orthogonalMichael addition reactions. Both tris(2-aminoethyl)amine andtetradecylamine quantitatively and selectively reacted with the acrylatefunctionality in AEMA after 24 hours in the presence of 5 mol % ofbutylated hydroxyltoluene (BHT) (to inhibit radical formation) at 50°C., while AEMA by itself remains unreacted under these conditions (FIGS.2 & 3). In the second orthogonal reaction (sulfa-Michael addition),dimethylphenylphosphine was required as a catalyst to achieve the finalproduct in low concentration (the lowest is 125 mM) or small scale (˜20mg on average) conditions as well as to achieve high conversion (100% by¹H NMR) so that the material can be used without purification forsubsequent testing or generation expansion (FIGS. 4 & 5). Some of thedendrimers were re-synthesized in larger scale and purified by flashchromatography before conducting in vivo studies.

Due to multiple delivery barriers, the potency of small RNA carriersthrough nanoencapsulation is influenced by various factors, includingpK_(a), topology/structure, and hydrophobicity (Siegwart et al., 2011;Jayaraman et al., 2012; Schaffert et al., 2011; Whitehead et al., 2014).To easily identify degradable dendrimers with high delivery potency, alibrary of G1DDs was designed with four zones: core binding—peripherystabilization (zone I), core binding—periphery binding (zone II), corestabilization—periphery stabilization (zone III), and corestabilization—periphery binding (zone IV) by chemically diversifyingcore-forming amines C and periphery-forming thiols P (FIGS. 1C & 1D). Inzones I and II, RNA binding was modulated by amines with one (1An) tosix (6An) initial branching centers (IBCs). Corresponding dendrimerstherefore contained one to six branches. In zones III and IV,stabilization of RNA-dendrimer NPs was mainly changed with differentlength of alkyl chains (1Hn and 2Hn). In zones II and IV, bindingcapability of aminothiols (SNn) was mainly modulated with differentamines while in zones I and III, stabilization was changed withalkylthiol (SCn) length and carboxyl- and hydroxyl-alkylthiols (SOn).The entire library of compounds was tested for the dendrimers' efficacy(FIG. 6).

Example 4: In Vitro G1DD Screening for siRNA Intracellular Delivery

Delivery carriers must overcome a series of extracellular andintracellular barriers to enable small RNAs to be active inside of tumorcells. G1DDs were identified which can mediate siRNA to overcome theintracellular barriers by screening of the 1,512 member G1DD library forthe ability to deliver siRNA in vitro to HeLa cells that stablyexpressed luciferase. G1DDs were formulated into nanoparticles (NPs)containing luciferase-targeting siRNA (siLuc) and the helper lipidscholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and lipidPEG2000 (Akinc et al., 2008; Semple et al., 2010). Intracellulardelivery potential was assessed by quantifying luciferase reduction andcell viability (FIGS. 7-9).

In order to extract SAR from the in vitro data, we utilized adendrimer-inspired tree analysis process (FIGS. 7B & 9B). Among the1,512 dendrimers, 88 mediated luciferase silencing of >50% and the hitrate of the whole library was 6%. When the hit rate of all four zones(I-IV) was analyzed, the hit rate of zone I was 10%, while those of zoneII, III, and IV were 0%, 2%, and 3%, respectively. This result indicatedthat these dendrimers with siRNA-binding core and stabilizing periphery(zone I) have much higher intracellular siRNA delivery potential. Withinthe branching types of zone 1, the hit rates of dendrimers with an SOperiphery is as low as 1% while that of the dendrimers with an SCperiphery was as high as 15%. Without wishing to be bound by any theory,it is believed that the hydrophobic stabilization from the dendrimerperiphery is crucial to efficiently deliver siRNA into cells throughnanoencapsulation. This likely results in increased hydrophobic packingthat provides additional NP stability (Leung et al., 2012). After branchnumber and branch lengths of these dendrimers with binding core and SCperiphery was further examined, the dendrimers with binding core andthree, four, five or six SC5-8 branches or SC9-12 branches have >25%chance to deliver siLuc into HeLa cells with >50% luciferase knockdown.Through in vitro screening of the full G1DD library and the dendriticanalysis process, the group of dendrimers which showed increasedintracellular siRNA delivery was identified: the groups with bindingcore/SC periphery and binding core with three to six SC5-8 or SC9-12branches.

Example 5: Identification of Degradable Dendrimers for Effective In VivosiRNA Delivery and Design of G2-G4 Dendrimers

Having identified dendrimers that can overcome intracellular barriers,next, the dendrimers that can overcome extracellular barriers toefficiently deliver siRNA in vivo were identified. By separating thesetwo processes, chemical functionality that overcomes barriers includingblood stability, liver (tumor) localization, cellular uptake, and activesiRNA release could be identified. Dendrimers were evaluated for theirability to silence Factor VII in hepatocytes because this blood clottingfactor can be readily quantified from a small serum sample (Akinc etal., 2008; Semple et al., 2010). 26 of the hit degradable dendrimerswere selected to maximize chemical diversity: 22 possessed an optimizedchemical structure based on the dendritic analysis process and anadditional 4 (2A2-SC14, 2A6-SC14, 2A9-SC14, and 6A1-SO9) were chosenbased on their high intracellular siRNA delivery ability. Dendrimerswere formulated with anti-Factor VII siRNA (siFVII) and were injectedi.v. into mice at a dosage of 1 mg siFVII/kg. FVII activity wasquantified 3 days post injection. Despite high in vitro potency,2A2-SC14, 2A6-SC14, 2A9-SC14, 6A1-SO9 and most three-branch dendrimersshowed only minimal in vivo FVII knockdown (FIG. 3A). The dendrimersthat contained a binding core and four, five or six SC8 or SC12 branchesshowed higher knockdown. Based upon these studies, SC8 branch dendrimerswere generally more effective than SC12 branch compounds.

With the in vitro and in vivo high-throughput screening results in hand,we asked whether we could now use that SAR information to rationallydesign dendrimers with predicted activity to validate our approach. Aseries of degradable dendrimers were prepared using two strategies: (I)by choosing polyamines with five or six IBCs; and (II) by increasingbranches via dendrimer generation expansion (FIG. 10). Two naturalamines, spermidine (5 IBCs) and spermine (6 IBCs), were chosen toimplement strategy I. As to strategy II, 1A2 (one IBC), 2A2 & 2A11 (twoIBCs), 3A3 & 3A5 (three IBCs), and 4A1 & 4A3 (four IBCs) were chosen toyield degradable dendrimers with multiple branches via generationexpansion (FIGS. 10C & 11). 24 additional degradable dendrimers wereevaluated (FIG. 10C) to further examine in vivo SAR. After generationexpansion, higher-generation dendrimers of 1A2 (one IBC), 2A2 (twoIBCs), and 3A3 & 3A5 (three IBCs) with four or six SC branches had goodin vivo siRNA delivery to hepatocytes, while the dendrimers with eightbranches were less active. This process transformed amine cores thatwere inactive in the in vitro screen, and then rationally design highergeneration dendrimers which showed in vivo activity.

Example 6: In Vivo Toxicity Evaluation of Degradable Dendrimers in MiceBearing MYC-Driven Liver Tumors

To identify degradable dendrimers with the required balance of lowtoxicity and high potency required for liver cancer treatment, thedegradable dendrimers were chose to evaluate their in vivo toxicity. Inparallel, we analyzed C12-200 lipidoid LNPs wee chose as the bestexample of a non-hydrolyzable system previously shown to be potent inmice and non-human primates (Love et al., 2010). Lipidoids, as a class,are benchmark materials at the forefront of clinical research (Kanastyet al., 2013; Love et al., 2010; Sahay et al., 2013). Non-immunogeniccontrol siRNA was used to best evaluate the toxicity of the individualdendrimers themselves. Dendrimer NPs were formulated at a weight ratioof 25:1 (dendrimer:siCTR), higher than necessary to better probetoxicity. C12-200 LNPs were prepared using identical formulationparameters as previously reported (Love et al., 2010). Size and zetapotential of each NP in PBS buffer was characterized. They all possessedsimilar size, 64-80 nm in diameter, and their surfaces were close toneutral in charge (FIGS. 12A & 12B). Each formulated NP was injectedi.v. into wild type mice at a 4 mg siCTR/kg dose (100 mg dendrimer/kg or28 mg C12-200/kg). Among the many different ways to evaluate in vivotoxicity, body weight loss can be utilized as a simple and informativeparameter. In normal mice, there were minimal body weight changes forthe selected NPs, including C12-200 control LNPs. However, amongcandidates, the mice injected with 5A2-SC8 and 6A3-SC12 experiencedquicker recovery and gained weight normally after the first day.

Based on these results, 5A2-SC8 and 6A3-SC12 were chose for furtherevaluation of their in vivo toxicity in chronically ill transgenic micebearing aggressive liver tumors with single and multiple injections. Awell-established Tet-On MYC inducible transgenic liver cancer model waschosen (FIG. 13A) (Nguyen et al., 2014). Since tumors are moreaggressive when MYC is overexpressed at earlier developmental timepoints, MYC was induced immediately after birth (p0), which resulted inrapidly growing liver tumors. At the age of 32 days (p32), these sicktransgenic mice bearing aggressive liver tumors were injected with5A2-SC8 or 6A3-SC12 NPs at 3 mg siCTR/kg dosage (75 mg dendrimer/kg or21 mg C12-200/kg). The mice receiving 5A2-SC8 injection lost about 5%body weight on the first day and quickly returned to their startingweight on the second day while those mice receiving 6A3-SC12 injectionstill lost 10% body weight by the third day and could not recover (FIG.12D). After multiple injections, these mice died seven days earliercompared to mice that received no treatment because of the toxicity of6A3-SC12 carrier (FIG. 12E). In contrast to the result in WT mice,injection of C12-200 LNPs to mice bearing aggressive tumors lost >20%weight after one day, despite receiving ˜3 times less lipid than 5A2-SC8injected mice (FIG. 12D). These data showed that small changes inchemical structure can produce large changes in toxicity. It also showedthat tumor-bearing mice are more sensitive to intervention than healthymice. Based on these results, 5A2-SC8 emerged as a degradable dendrimerpossessing a balance of low toxicity (tolerance in tumor-bearing mice upto 75 mg/kg) and effective in vivo FVII knockdown (>95% at 1 mgsiFVII/kg). In addition to being less toxic than benchmark compounds,5A2-SC8 NPs are more efficacious because these dendrimers reduceclinical concern for dose limiting toxicity and enable a widertherapeutic window.

Example 7: Potent Suppression of Liver Tumor Growth Through SystemicAdministration of a Let-7g miRNA Mimic

In order to evaluate the ability of degradable dendrimer NPs to delivera therapeutic miRNA mimic without causing additional toxicity, theaggressive, MYC transgenic liver cancer model induced at p0 was againused (Nguyen et al., 2014). These mice developed rapidly growing cancersresembling pediatric hepatoblastoma (HB), a tumor type that shares manyof the molecular features of HCC. Abdominal distention from mass effectwas grossly visible after 20 days, and tumors grew rapidly. Withoutintervention, mice died within 60 days after birth. Given the speed andlethality of this model, there are limited opportunities for successfultherapy.

Since 5A2-SC8 balances low in vivo toxicity and effectiveness forsilencing the hepatocellular target FVII, first, whether 5A2-SC8 NPscould deliver siRNA into tumor cells was examined. At the age of 41 days(p41), the livers of these transgenic mice are full of tumors (FIG.14A). On p40, mice were injected intravenously with 5A2-SC8 NPs withCy5.5-labeled siRNA at a dosage of 1 mg siRNA/kg. 24 hourspost-injection, fluorescence imaging showed 5A2-SC8 mediated siRNAaccumulation in the cancerous liver, with only minor accumulation in thespleen and kidneys (FIGS. 14A-14B). 5A2-SC8 NPs delivered siRNAs intonormal and transgenic livers even if the cancerous livers are largerthan normal ones (FIG. 15A).

To further confirm whether 5A2-SC8 NPs can deliver siRNA in vivo intotumor cells, tumor tissues of the liver were collected and imaged 24hours after intravenous injection. H&E staining showed the tumor tissuesare densely full of cellular nuclei and exhibit a cancerous phenotype(FIG. 15B). Confocal imaging confirmed that 5A2-SC8 NPs were able toeffectively deliver labeled siRNA into tumor cells (FIG. 14C).

The therapeutic benefit of 5A2-SC8-mediated small RNA delivery in thesechronically ill transgenic mice was then evaluated. One of the mostimportant miRNAs is Let-7, a tumor suppressor family downregulated inmany tumor types (Boyerinas et al., 2010; Roush and Slack, 2008).Because endogenous Let-7g is known to be downregulated in liver HB(Nguyen et al., 2014), tests were conducted to determine if delivery ofa Let-7g mimic in this aggressive, genetically engineered mouse modelcould inhibit the development of liver cancer.

The 5A2-SC8 NPs were verified to be able to enable siRNA delivery inthis model. Delivery of a single dose of siFVII i.v. showed potentsilencing of FVII protein using a blood assay (FIG. 16A) and by qPCR inharvested liver tissues (FIG. 16B). This silencing was achieved on p26,which is after tumor development has initiated. Next, 1 mg/kg Let-7g wasdelivered in 5A2-SC8 NPs i.v. to tumor-bearing mice (p26). Let-7gexpression was increased 7-fold in liver tissues 48 hours post-injection(FIG. 16C).

Then, a therapeutic regimen from p26 by weekly administration of 5A2-SC8NPs containing Let-7g mimic or Control mimic at 1 mg/kg was started. Atp50, the mice that received the Let-7g mimic had grossly smallerabdomens and reduced tumor burden (FIGS. 16D-16F). Let-7g causedreduction of abdominal circumference, quantitative of tumor growth (FIG.16E). The effect on tumor growth was confirmed by ex vivo liver imaging(FIG. 16F). Most importantly, delivery of Let-7g weekly from 26 to 61days did not affect weight gain (FIG. 16G) and significantly extendedsurvival (FIG. 16H). All control mice receiving no treatment and micereceiving 5A2-SC8 NPs with CTR-mimic died around 60 days of age. C12-200LNPs (Let-7g or control mimic) induced premature death, and requiredhalting of the experiment. Delivery of Let-7g inside of 5A2-SC8 NPsprovided a dramatic survival benefit, with one mouse living to 100 days.These results showed that 5A2-SC8 can balance high delivery efficacywith low toxicity to provide a significant therapeutic benefit tochronically ill transgenic mice by effective inhibition of liver tumorgrowth.

Example 8: Evaluation of Different Lipid Compositions for siRNA Delivery

To evaluate which lipid composition within the dendrimer nanoparticleslead to improved siRNA delivery, the identity and concentration ofdifferent phospholipids and PEG-lipids were varied. Three different celllines (HeLa-Luc, A549-Luc, and MDA-MB231-Luc) were used. The cells werepresent at 10K cells per well and a 24 hour incubation. The readout wasdetermined 24 hours post transfection. In the nanoparticles, DSPC andDOPE were used as phospholipids and PEG-DSPE, PEG-DMG, and PEG-DHD wereused as PEG-lipids. The compositions contain a lipid ordendrimer:cholesterol:phospholipid:PEG-lipid mole ratio of 50:38:10:2.The mole ratio of lipid/dendrimer to siRNA was 100:1 with 100 ng dosebeing used. The RiboGreen, Cell-titer Fluor, and OneGlo assays were usedto determine the effectiveness of these compositions. Results show therelative luciferase activity in HeLa-Luc cells (FIG. 17A), A549-Luc(FIG. 17B), and MDA-MB231-Luc (FIG. 17C). The six formulations used inthe studies include: dendrimer (lipid)+cholesterol+DSPC+PEG-DSPE(formulation 1), dendrimer (lipid)+cholesterol+DOPE+PEG-DSPE(formulation 2), dendrimer (lipid)+cholesterol+DSPC+PEG-DMG (formulation3), dendrimer (lipid)+cholesterol+DOPE+PEG-DMG (formulation 4),dendrimer (lipid)+cholesterol+DSPC+PEG-DSPE (formulation 5), anddendrimer (lipid)+cholesterol+DOPE+PEG-DHD (formulation 6).

Further experiments were run to determine which phospholipids showed theincreased delivery of siRNA molecules. A HeLa-Luc cell line was usedwith 10K cells per well, 24 hour incubation, and readout 24 hours posttransfections. The compositions contained either DOPE or DOPC as thephospholipid with PEG-DHD as the PEG-lipid. The ratio of lipid (ordendrimer):cholesterol:phospholipid:PEG-lipid was 50:38:10:2 in a moleratio with the mole ratio of dendrimer (or lipid) to siRNA of 200:1.These compositions was tested at a 50 ng dose using the Cell-titer Fluorand OneGlo assays. These results are shown in FIGS. 18A & 18B.

Example 9: Evaluation of Dendrimer Nanoparticles for Delivery of sgRNAand Other CRISPR Nucleic Acids

To evaluate the compositions to delivery nucleic acids for CRISPR/Casgene editing, the delivery of sgRNA and mRNA was tested. Cell lines werecreated that could allow for rapid screening of dendrimer NPs and Z120for sgRNA delivery. For example, HeLa (cervical cancer) and A549 (lungcancer) cells were established to co-express luciferase and Cas9.Selection and quality control was verified. Guide RNAs were designedaccording to previously reported methods targeting the first exon of thedesired target gene. Targets possessing the highest score indicatingcleavage activity and sequence specificity were carried forward forsgRNA preparation using established protocols. DNA oligonucleotides weresynthesized commercially, annealed, cloned by BsbI digestion and ligatedinto a plasmid backbone containing Cas9. In vitro transcription enabledthe isolation of sgRNA, which could then be packaged into dendrimer NPsfor delivery. A series of 5 different guides were designed forLuciferase. These guides were validated by sgLuc-Cas9 pDNA transfectionusing commercial reagents to select the best sgRNA sequence. Next, wepackaged sgLuc into dendrimer NPs and evaluated delivery inHeLa-Luc-Cas9 cells for delivery of sgRNA. Following a determined numberof hours of exposure, luciferase and viability were measured compared tountreated cells using One Glo+Tox (Promega). In a typical experiment,10K cells were plated per well, followed by 24 hour incubation, additionof dendrimer nanoparticles containing sgLuc, and readout at 24-48 hourspost transfection. These compositions contained combinations ofdendrimers, DSPC or DOPE, cholesterol, and PEG-lipid. Additionally, thecompositions contained various concentrations of MgCl₂. Molar ratios oflipid (or dendrimer):cholesterol:PEG-lipid were 50:38.5:0.5 with a moleratio of lipid to nucleic acid (sgRNA) of 200:1 and a 50 ng dose. Again,the Cell-titer Fluor and OneGlo assays were used to obtain the results.Results without a phospholipid are shown in FIG. 19. Similar studieswere carried out with phospholipid present. In these compositions, thephospholipid DSPC was used in the formulations. Using the same ratio asabove for compositions which did not contain a phospholipid, thephospholipid containing compositions had a mole ratio of 50:38.5:10:0.5(lipid/dendrimer:cholesterol:phospholipid:PEG-lipid) using the samedosing amount. These compositions were tested using RiboGreen,Cell-titer Fluor, and OneGlo at two time periods, 24 hours and 72 hours.Data obtained at 24 hours is shown in FIG. 20A and 72 hours is shown inFIG. 20B.

Example 10: Evaluation of Dendrimer Nanoparticles for Delivery of mRNA

Similar, to the studies carried out with siRNA, the delivery of mRNAmolecules were tested with the dendrimers described herein and Z120. AIGROV1 cell line was used at a concentration of 4K cells per well, 24hour incubation, and readout at 24 hours and 48 hours post transfection.These compositions contained either DSPC, DOPE, or no phospholipid andPEG-DHD as the PEG-lipid. Molar ratios of lipid (ordendrimer):cholesterol:phospholipid:PEG-lipid were 50:38.5:0(10):2 witha weight ratio of dendrimer to nucleic acid (mRNA) of 5, 10, 20, 30, or40 to 1 and two different doses: a 50 ng dose and a 100 ng dose. TheCell-titer Fluor and OneGlo assays were used to obtain the results.These results were shown are shown in FIG. 21A (24 hours) and FIG. 21B(48 hours). Additionally, the delivery of mCherry mRNA into wasvisualized in FIG. 22 using a nanoparticle composition with 20:1 ratioN/P and DSPC as the phospholipid and PEG-DHD as the PEG-lipid.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of certain embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe methods described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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What is claimed is:
 1. A method for modulating an amount or activity ofa gene or a gene product in a cell, the method comprising contactingsaid cell with said therapeutic agent assembled with a lipidcomposition, which lipid composition comprises a dendrimer or dendroncomprising one or more degradable diacyl group, thereby modulating saidamount or activity of said gene or said gene product in said cell. 2.The method according to claim 1, wherein said therapeutic agentcomprises a nucleic acid.
 3. The method according to claim 2, whereinsaid nucleic acid comprises siRNA, a miRNA, a pri-miRNA, a messenger RNA(mRNA), a cluster regularly interspaced short palindromic repeats(CRISPR) related nucleic acid, a single guide RNA (sgRNA), a CRISPR-RNA(crRNA), a trans-activating crRNA (tracrRNA), a plasmid DNA (pDNA), atransfer RNA (tRNA), nan antisense oligonucleotide (ASO), a guide RNA, adouble stranded DNA (dsDNA), a single stranded DNA (ssDNA), a singlestranded RNA (ssRNA), or a double stranded RNA (dsRNA).
 4. The methodaccording to claim 1, wherein said contacting comprises administeringsaid therapeutic agent assembled with said lipid composition to asubject comprising said cell.
 5. The method according to claim 4,wherein said administering comprises administering orally,intraadiposally, intraarterially, intraarticularly, intracranially,intradermally, intralesionally, intramuscularly, intranasally,intraocularly, intrapericardially, intraperitoneally, intrapleurally,intraprostatically, intrarectally, intrathecally, intratracheally,intratumorally, intraumbilically, intravaginally, intravenously,intravesicularlly, intravitreally, liposomally, mucosally, parenterally,rectally, subconjunctival, subcutaneously, sublingually, topically,transbuccally, transdermally, or vaginally.
 6. The method according toclaim 4, wherein said modulating is sufficient to treat a disease ordisorder in said subject.
 7. The method according to claim 1, whereinsaid dendrimer or dendron is present in said lipid composition at aweight ratio to said nucleic acid of about 100:1 to about 1:5.
 8. Themethod according to claim 1, wherein said lipid composition furthercomprises a steroid or steroid derivative, a polymer-conjugated lipid, aphospholipid, or a combination thereof.
 9. The method according to claim8, wherein said steroid or steroid derivative is present in said lipidcomposition at a molar ratio to said dendrimer or said dendron of about10:1 to about 1:20.
 10. The method according to claim 8, wherein saidpolymer-conjugated lipid is present in said lipid composition at a molarratio to said dendrimer or dendron of about 1:1 to about 1:250.
 11. Themethod according to claim 8, wherein said phospholipid is present insaid lipid composition at a molar ratio to said dendrimer or dendron ofabout 10:1 to 1:20.
 12. A composition for modulating an amount oractivity of a gene or a gene product in a cell, the compositioncomprising a therapeutic agent assembled with a lipid composition, whichlipid composition comprises a dendrimer or a dendron comprising one ormore degradable diacyl group.
 13. The composition according to claim 12,wherein said therapeutic agent comprises a nucleic acid.
 14. Thecomposition according to claim 13, wherein said nucleic acid comprisessiRNA, a miRNA, a pri-miRNA, a messenger RNA (mRNA), a cluster regularlyinterspaced short palindromic repeats (CRISPR) related nucleic acid, asingle guide RNA (sgRNA), a CRISPR-RNA (crRNA), a trans-activating crRNA(tracrRNA), a plasmid DNA (pDNA), a transfer RNA (tRNA), nan antisenseoligonucleotide (ASO), a guide RNA, a double stranded DNA (dsDNA), asingle stranded DNA (ssDNA), a single stranded RNA (ssRNA), or a doublestranded RNA (dsRNA).
 15. The composition according to claim 19, whereinsaid lipid composition is formulated for administration: orally,intraadiposally, intraarterially, intraarticularly, intracranially,intradermally, intralesionally, intramuscularly, intranasally,intraocularly, intrapericardially, intraperitoneally, intrapleurally,intraprostatically, intrarectally, intrathecally, intratracheally,intratumorally, intraumbilically, intravaginally, intravenously,intravesicularlly, intravitreally, liposomally, mucosally, parenterally,rectally, subconjunctival, subcutaneously, sublingually, topically,transbuccally, transdermally, or vaginally.
 16. The compositionaccording to claim 19, wherein said dendrimer or dendron is present insaid lipid composition at a weight ratio to said nucleic acid of about100:1 to about 1:5.
 17. The composition according to claim 16, whereinsaid composition further comprises a steroid or steroid derivative, apolymer-conjugated lipid, a phospholipid, or a combination thereof. 18.The composition according to claim 17, wherein said steroid or steroidderivative is present in said lipid composition at a molar ratio to saiddendrimer or said dendron of about 10:1 to about 1:20.
 19. Thecomposition according to claim 17, wherein said polymer-conjugated lipidis present in said lipid composition at a molar ratio to said dendrimeror dendron of about 1:1 to about 1:250.
 20. The composition according toclaim 17, wherein said phospholipid is present in said lipid compositionat a molar ratio to said dendrimer or dendron of about 10:1 to 1:20.